FROM  THE  LIBRARY  OF 
WILLIAM  A.  SETCHELL,i864-i943 

PROFESSOR  OF  BOTANY 


PLATE  I. 


'*:: 


TEST    OBJECTS 


PLATE  II, 


TEST   OBJECTS 


n 


A    GUIDE 


FOR  THE 


MICROSCOPICAL  INVESTIGATION 


OF 


VEGETABLE  SUBSTANCES 

FROM  THE  GERMAN  OF 

DR.   JULIUS   WILHELM   BEHRENS 

\\ 

TRANSLATED  AND  EDITED  BY 

REV.  A.  B.  HERVEY,  A.M. 

ASSISTED   BY 

R.  H.  WARD,  M.D.,  F.R.M.S. 


ILLUSTRATED 

WITH  THIRTEEN  PLATES  AND  ONE  HUNDRED  AND  FIFTY-THREE  CUTS 


Bo0ton 

S.   E.    CASSINO   AND   COMPANY 
1885 


COPYRIGHT  1885 
BY 

S.  E.  CASS1NO  &  CO. 


BIOLOGY  LIBRARY 


- 

K 

B 


i 


AUTHOR'S  PREFACE. 


THE  preparation  of  this  work  has  engaged  my  time  for  sev- 
eral years.  In  the  beginning  of  1880,  I  finally  concluded  to 
work  up  for  publication  the  material  relating  to  the  microscop- 
ical investigation  of  vegetable  substances,  which  I  had  previously 
collected  for  private  use.  Several  friendly  botanists,  to  whom 
I  communicated  my  designs,  counseled  me  very  earnestly  to 
carry  them  out.  As  the  publishers  also  were  prepared  to  un- 
dertake the  work  at  once,  the  preparation  of  the  manuscript  and 
the  printing  of  it  have  gone  on  simultaneously  since  about 
Easter,  1880. 

For  a  work  to  be  useful  in  those  microscopical  inquiries 
which  are  most  important  in  the  botanical  laboratory,  it  need 
teach  neither  optics  nor  histology. 

The  student  will,  therefore,  find  in  the  work  before  him  a 
brief  description  only,  of  the  microscopical  apparatus  applicable 
to  his  uses  (Chapters  I  and  II),  together  with  directions  for 
its  use.  If  he  wishes  to  become  acquainted  with  the  instru- 
ment from  the  standpoint  of  the  optical  physicist  he  must  go 
to  the  larger  manuals  of  Hartig,  of  Nageli  and  Schwendener, 
or  the  shortly  to  be  published  hand-book  of  Dippel  and  Abbe. 
These  are  works  which,  if  studied  with  the  necessary  care,  will 
furnish  a  very  perfect  understanding  of  the  performance  of  the 
microscopical  apparatus,  but  which  on  account  of  their  lengthy 
theoretical  analyses  are  but  poorly  adapted  for  the  table  of  the 
practical  microscopist. 

The  first  and  second  chapters  treat  of  the  microscope  and  its 
accessory  apparatus,  while  the  third  contains  directions  for  the 
preparation  of  microscopic  specimens.  Every  one  knows 
that  the  preparation  of  specimens  cannot  be  learned  by  the  mere 


iv  AUTHOR'S  PREFACE. 

reading  of  these  detailed  statements.  In  this  matter  manual 
instruction  is  the  main  thing.  However,  the  study  of  this  chap- 
ter will  open  in  many  places  new  points  of  view  to  the  youn^ 
.microscopist,  and  give  him  occasion  here  and  there  independ- 
ently to  apply  new  methods. 

I  have  to  thank  my  friend,  Dr.  Conwentz,  for  kindly  under- 
taking the  preparation  of  the  section  relating  to  fossil  plants. 

The  most  important  part  of  the  whole  work  is  in  the  fourth 
and  fifth  chapters.  They  contain  what  has  heretofore  —  incor- 
rectly—  been  called  micro-chemistry.  The  fourth  chapter 
treats  of  microscopical  reagents  and  the  fifth  of  the  microscop- 
ical investigation  of  vegetable  substances. 

Until  the  middle  of  1880,  there  was  no  useful  compilation 
of  the  matters  pertaining  to  this  subject  which  was  at  all  abreast 
•with  the  science  of  the  day  in  existence.  But  in  the  meantime 
.the  "Botanical  Micro-chemistry"  of  Poulsen,  a  brief  compilation 
of  the  methods  of  micro-chemical  reactions,  has  appeared. 
I  believe,  however,  that  notwithstanding  this  little  book  has 
justly  had  a  wide  circulation,  the  value  of  the  corresponding 
chapters  of  the  present  work  will  not  be  materially  lessened. 

Poulsen's  work  is  designed  mainly  for  beginners  and  there- 
fore contains  only  the  most  important  methods  of  reaction  in 
the  barest  outline.  On  the  other  hand,  the  chapter  of  this 
work  which  deals  with  the  microscopical  investigation  of  vege- 
table substances,  furnishes  an  exhaustive  treatment  of  these 
matters,  and  at  the  same  time  is  so  arranged  as  to  make  the 
specialist  quite  independent  of  the  widely  dispersed  literature 
of  the  subject  which  is  often  hidden  and  not  seldom  difficult  to 
obtain.  But  at  the  same  time,  a  compilation  of  the  literature, 
as  complete  as  possible,  greatly  facilitates  reference  to  the  orig- 
inal works.  It  is  evident  that  the  point  of  view  thus  briefly 
outlined  must  require  a  handling  of  the  case  fundamentally  differ- 
ent from  that  which  Poulsen  has  given  it.  I  have  kept  the 
chemical  (i.  e.,  the  physio logico-chemical)  point  of  view  in  the 
foreground  throughout,  not  only  in  the  arrangement  of  the  whole, 
but  also  in  the  management  of  each  separate  section.  The 
arrangement  of  the  subject  matter  follows  closely  that  of  the 


AUTHOR'S   PREFACE.  v 

new  edition  of  Husemann  and  Hilger's  "Vegetable  Substances," 
("Pflanzenstoflfe")  which  I  regret  to  say  is  not  vet  completed. 
Thus  the  use  of  that  work  in  connection  with  my  compilation 
is  made  more  convenient.  I  am  firmly  convinced  that  the  mi- 
cro-chemist will  have  many  interesting  outlooks  opened  to  him, 
and  many  new  methods  suggested  by  the  study  of  Husemann 
and  Hilger's  work.  It  seems  to  me  that  the  separate  micro- 
scopical investigation  of  vegetable  substances  is  the  only  way 
(leading  out  from  their  chemical  qualities)  to  attain  a  true 
comprehension  of  the  methods  of  microscopical  research. 

The  discerning  reader  will  soon  discover  that  the  whole  chap- 
ter is  by  no  means  a  mere  compilation,  but  that  I  have  critically 
sifted  the  existing  materials.  The  useless  I  have  rejected. 
The  useful,  however,  I  have  taken  not  altogether  on  trust  and 
faith,  but  as  far  as  possible  carefully  tested.  Indeed,  I  have 
tested  everything  it  was  possible  to  in  the  nature  of  things,  and 
this  experimentation  has  already  consumed  more  than  three 
years  of  working  time. 

For  the  presentation  of  the  whole  I  have  chosen  —  as  brevity 
also  seemed  to  require  —  a  purely  objective  form.  Subjective 
views  are  kept  entirely  in  the  background,  and  at  no  time 
have  I  entered  into  argument.  Various  new  discoveries,  the 
results  of  my  experimentations,  will  be  published  later  in  a 
separate  monograph. 

In  the  compilation  of  the  literature  I  have  attempted  the  ut- 
most possible  completeness.  For  my  success  in  this,  I  am 
mainly  indebted  to  the  University  library,  which  lacks  scarcely 
a  single  treatise  of  all  the  literature  quoted.  With  hardly  a 
noteworthy  exception,  I  have  seen  and  read  it  all. 

But  a  small  portion  of  the  illustrations  for  this  work  are  cop- 
ies. Much  the  greater  part  are  original  drawings  which  I  have 
for  the  most  part  made  upon  wood  myself.  So  far  as  this  re- 
mark applies  to  the  microscopical  apparatus,  they  have  been 
photographed  under  my  directions  with  the  use  of  a  very  small 
diaphragm ;  they  appeared  almost  black,  but  for  engraving,  I 
finished  the  pictures,  which  bad  been  photographically  trans- 
ferred to  the  wood,  in  lines  with  sepia  or  India  ink  and  white. 


vi  AUTHOR'S  PREFACE. 

Since  the  printing  went  on  continuously  with  the  elaboration 
of  the  manuscript,  I  could  not  in  some  sections  refer  to  the  very 
latest  literature,  as,  for  example,  in  Cellulose,  Strasburger's 
beautiful  work  on  the  structure  and  growth  of  the  cell  wall 
could  not  be  cited,  likewise  some  recent  monographs  concern- 
ing the  structure  of  the  nucleus.  But  as  far  as  it  was  possible 
I  have  cited  the  very  latest  literature. 

I  shall  be  much  obliged  to  those  gentlemen  who  may  give 
the  fifth  chapter  critical  attention  if  they  will  communicate  to 
me  any  accidental  omission  or  error. 

W.  BEHRENS. 
GiMingen,  Dec.  18,  1882. 


TRANSLATOR'S  PREFACE. 


IN  presenting  to  the  English-speaking  public  a  translation"^ 
Dr.  Behrens'  invaluable  work,  a  few  explanatory  words  seem 
to  be  needed. 

It  is  the  first  purpose  of  this  work  to  guide  students  in  all 
those  inquiries  relating  to  the  physical  products  of  cell-life  in 
plants,  which  may  be  conducted  under  the  microscope,  by 
means  of  chemical  and  other  reactions.  It  undertakes  so  to  in- 
struct him  that  he  can  make  a  thin  section  of  any  part  or  organ 
of  a  plant,  and,  putting  it  under  his  lens,  answer  to  himself  the 
questions  :  What  have  the  life  processes  thus  far  produced  here, 
and  what  are  they  now  producing? 

It  deals  with  the  anatomical  constitution  of  the  cell,  and  of 
plant  tissue,  and  yet,  its  inquiries  relate  far  more  to  physiolog- 
ical and  biological  processes  and  results  than  to  matters  purely 
anatomical  and  histologies!.  The  unit  of  life  is  the  cell.  The 
physical  embodiment  of  that  life  is  the  protoplasm,  the  physio- 
logical center  of  which  is  the  nucleus.  The  formed  matter,  the 
finished  product  of  life,  is  the  cell  wall  and  some  cell  contents, 
and  in  the  tissue  the  middle  lamella  also.  The  life  of  different 
cells  and  its  embodiment  are  absolutely  indistinguishable.  The 
protoplasm  and  nucleus  of,  for  example,  wood-forming  cells  and 
cork-forming  cells  are  microscopically  and  chemically  alike.  Dif- 
ferentiation appears  only  in  the  finished  product  of  the  life  pro- 
cess, viz. :  in  some  cell-contents,  and  mainly  in  the  cell  wall.  It  is 
to  the  nature  of  this,  therefore,  largely,  in  all  the  various  kinds 
of  vegetable  tissue  that  our  inquiries  relate.  But  not  alone  to 
this  :  for  a  full  investigation  of  nearty  all  the  other  elements  of 
plant  life  is  carefully  marked  out, — of  functional  protoplasm  and 
reserve  protoplasm  or  proteid  matter,  of  starch,  chlorophyll, 
sugar,  etc.,  elements  so  largely  concerned  in  the  life  processes. 

The  treatise  occupies  a  field  almost  entirely  to  itself  in  the  bo- 
tanical literature  both  of  Germany,  and    now  of  the  English- 

(vii) 


viii  TRANSLATOR'S   PREFACE. 

speaking  world.  It  is  sincerely  hoped  that  its  publication  in 
this  form  will  stimulate  in  this  country  investigations  into  the 
deeper  problems  of  plant  life.  It  will  be  seen  by  reference  to 
the  literature  cited,  that  there  is  an  open  field  for  American  bot- 
anists, since  the  works  referred  to  almost  exclusively  embody  the 
results  of  German  research,  while  a  few  are  of  French  origin, 
fewer  still  of  English  and  none  whatever  of  American. 

I  alone  am  responsible  for  the  translation,  and  have  endeav- 
ored to  take  a  medium  course  in  it,  following  the  text  neither 
too  literally,  nor  yet  translating  so  freely  as  to  introduce  shades 
of  meaning  not  in  the  author's  mind.  1  am  inclined  to  believe 
my  errors  are  those  more  often  leaning  to  the  former  than  to 
the  latter  side.  In  that  direction,  I  suppose,  lie  the  tempta- 
tions for  the  conscientious  translator,  though  in  the  interests  of 
a  good  style  he  would  be  more  readily  pardoned  for  sinning  on 
the  other  side. 

Early  in  the  enterprise,  Dr.  K.  H.  Ward  very  kindly  con- 
sented to  undertake  the  revision  of  the  two  chapters  which  deal 
with  the  microscope  and  its  accessories.  He  states  the  plan 
upon  which  he  has  made  the  somewhat  extensive  changes  in 
these  chapters  as  follows  :  — 

"  The  changes  in  Chapters  I  and  II  consist  wholly  in  the  omis- 
sion of  illustrations  and  descriptions  of  apparatus  in  the  Conti- 
nental style,  which  is  comparatively  unused  and  unavailable 
here,  and  the  substitution  of  American  forms.  It  was  desired, 
and  deemed  necessary,  that  a  work  intended  as  a  practical  hand- 
book should  describe  and  discuss  such  instruments  as  are  likely 
to  be  most  generally  preferred  and  used  by  the  majority  of  its 
readers.  The  author's  full  and  very  valuable  discussions  on  the 
methods  of  work,  and  on  the  construction,  testing,  care  and  use 
of  the  optical  parts,  and  of  the  most  important  accessories,  are 
retained  without  abridgment  or  material  amendment. 

"The  construction  of  the  Stand  is  illustrated  by  several  models, 
in  order  to  exhibit  the  most  common  varieties  believed  to  be 
eligible  for  such  work,  and  (incidentally)  to  establish,  by  com- 
parison of  characteristic  stands  by  prominent  American  makers, 
the  claim  to  the  existence  of  a  new,  serviceable  and  American 
type  in  addition  to  the  two  styles,  Continental  and  English, 


TRANSLATOR'S   PREFACE.  ix 

heretofore  recognized.  It  will  be  noticed  that  the  American 
type,  as  illustrated  by  Plates  III  to  VI,  Villa,  and  IX  to  XI, 
is  intermediate  in  size  and  complexity  between  the  other  two, 
but  built  upon  a  radically  different  model  from  either ;  having 
much  of  the  simplicity  and  portability  of  the  Continental  with 
much  of  the  efficiency  and  versatility  of  the  English  style." 

The  changes  in  the  third,  fourth  and  fifth  chapters,  for  which 
I  alone  am  responsible,  consist  almost  entirely  of  additions  to 
the  text.  They  relate  in  the  third  chapter  exclusively  to  those 
methods,  tools,  materials,  etc.,  which  experience  has  taught 
American  investigators  to  consider  important  in  their  work. 
The  few  additions  to  the  remaining  chapters  relate  only  to  those 
researches  in  this  field,  which,  up  to  the  beginning  of  1884, 
and  subsequent  to  the  close  of  the  author's  work,  had  come 
under  my  notice. 

The  matter  introduced  into  the  text  by  the  American  editors 
is  inclosed  in  brackets  [  ]  and  usually  signed  with  the  respec- 
tive editors'  initials.  Foot-notes  by  either  of  the  editors  are 
likewise  mostly  signed,  and  are  referred  to  in  the  text  by  sym- 
bols, while  those  of  the  author  are  all  numbered. 

We  are  indebted  to  various  firms  of  opticians,  as  well  as  to 
other  parties,  for  the  use  of  electrotypes,  for  which  we  here 
desire  to  express  our  cordial  thanks. 

The  work  of  translating  and  editing  this  treatise  has  been 
done  in  the  midst  of  the  engagements  of  a  busy  professional  life, 
in  hours  snatched  at  irregular  intervals  from  the  demands  of 
pressing  public  and  domestic  cares  ;  yet  I  have  verified  by  actual 
experiment  the  larger  part  of  all  the  statements  and  methods 
given  by  the  author. 

A.  B.  HEKVEY. 

Taunton,  Mass.,  Feb.  26,  1885. 


TABLE  OF  CONTENTS. 


CHAPTER  I.— THE  MICROSCOPE. 


I. 

Introduction,             

. 

II. 

The  Compound  Microscope,      

.       14 

I. 

The  Microscope  Stand,              ..... 

.       15 

A.     The  Student  Microscope,             .... 

.       16 

B.     The  Model  Microscope,       ..... 

17 

C.     The  Acme  Microscope,       ..... 

18 

D.     The  New  Student  Stand,            .... 

.       18 

E.     The  Physicians'  Stand,       

.       19 

F.     The  Continental  Student  Stand, 

.       19 

G.     The  Illustrator's  Stand,               .... 

.       20 

H.     The  Histological  Stand,             .... 

.       20 

I.     The  Biological  Stand,         .         . 

.       21 

J.     The  Universal  Stand,         ..... 

.       21 

II. 

The  Objective,          .                  

.       22 

A.     The  set  of  Achromatic  Lenses, 

.       23 

B.     The  Objective  System,         

.       23 

The  System  in  Practice,           .... 

.       28 

The  Immersion-System,           .... 

.       30 

The  Correction-System,            .... 

.       32 

III. 

The  Ocular,     

35 

IV. 

38 

V. 

The  Eye  Shade,         

.       42 

VI. 

Magnifying  power  of  the  Modern  Microscope, 

.       43 

VII. 

Testing  the  Optical  Powers,             .... 

.       53 

A.     Testing  the  Definition,       

.       54 

B.     Testing  the  Resolution,                .... 

.       57 

VIII.     The  Microscope-Tube,           ..... 

.       71 

IX. 

Nose-Pieces,             ....... 

.       74 

X. 

Fine  Adjustment,      ....... 

.       77 

XL 

The  Stage,      

.       80 

XII. 

The  Illuminating  Apparatus, 

.       82 

82 

B.     Diaphragms,       .         . 

.       84 

C.     Condensers,        ....... 

87 

(xi) 


xii  CONTENTS. 

D.  Illuminating  Combinations,         .....       90 

E.  Opaque  Illuminators,  ......       93 

F.  Observation  by  Artificial  Illumination,         ...       94 

XIII.  The  Microscope  Foot, 95 

XIV.  Rules  for  the  Use  of  the  Microscope,  .         .         .96 


CHAPTER  II.— MICROSCOPICAL  ACCESSORIES. 

I.  The  Preparing  Microscope, 100 

II.  Apparatus  for  drawing  Microscopic  Pictures,    .         .         .110 

III.  The  Micrometer  and  Microscopical  Measuring,        .         .120 

1.  Objective  Micrometers,  121 

2.  Ocular  Micrometer,  .         .         .         .         .         .122 

3.  Ocular  Screw  Micrometer, 126 

IV.  Camera  Lucida  as  a  Measuring  Apparatus,      .         .         .     127 

V.  Microscopic  Measuring  in  general,  .         .         .         .128 

VI.  Polarizing  Apparatus  and  Goniometer,             .         .         .133 
The  Goniometer,     . 137 

VII.  The  Micro-Spectroscope, 139 


CHAPTER  III.— PREPARATION    OF  MICROSCOPIC 
OBJECTS. 

I.  Introduction,  .         .         .         .         .         .         .         .156 

II.  Preparation  of  Objects  without  Cutting  Instruments,        .     160 

A.  Objects  for  immediate  Observation,    .         .         .  1 00 

B.  Macerating  or  Softening, 162 

C.  Incinerating  and  Calcining, 164 

III.  Instruments  for  preparing  Microscopic  thin  Sections,       .     1 65 

IV.  Cutting  Microscopical  Sections,       .         .         .         .         .177 

1.  Free  hand  cutting,  179 

2.  Cutting  by  means  of  Elder-pith  and  Cork,  .         .     183 

3.  Cutting  in  embedding  Media, 185 

4.  Cutting  sections  with  a  Microtome,  .         .         .     187 

V.  Further  treatment  of  the  section,  .         .         .         .196 

A.  Removing  the  Air,      .         .         .         .         .         .         .196 

B.  Handling  the  Section  under  the  preparing  Microscope,     197 

C.  Clarification  of  the  section,          .         .         .         .  198 

VI.  Preparation  of  Microscopic  specimens  of  Fossil  Plants,  .     203 


CONTENTS.  xiii 

VII.  Preparation  of  permanent  Mounts,         ....  213 

1.  Object-slide  and  Cover-glass, 214 

2.  Preserving  Media,* 217 

VIII.  Mounting  in  Preserving  Fluids,    .....  228 

IX.  Cementing  and  Finishing  the  Mount,       ....  234 

1.  Cements,     .........  234 

2.  Cementing  angular  Cover-glasses,        ....  237 

3.  Mounting  with  circular  Cover-glasses,          .         .         .  239 
Self-centering  Turn-tables,         ......  239 

X.  Labelling  and  Cataloguing  Preparations,  .         .         .  245 

XI.  Storing  permanent  Preparations, 247 

XII.  Examination  of  living  Organisms,  ....  249 

XIII.  Drawing  Microscopic  Objects,  ....  254 

1.  Aids  to  Microscopical  Drawing,  ....  254 

2.  Conducting  Microscopical  Drawing,  .         .         .  259 

3.  Drawing  Materials,     .......  265 


CHAPTER  IV.— MICROSCOPICAL  REAGENTS. 

I.  Introduction, 267 

II.  Apparatus  for  the  preparation  of  Reagents,       .         .         .272 

III.  The  Volumetric  Method, 278 

IV.  Enumeration  and  preparation  of  Microscopical  Reagents  283 
A.     Inorganic  Combinations,                 .....  283 

1.  Water, 283 

2.  Nitric  Acid, 284 

3.  Sulphuric  Acid, 284 

4.  Hydrochloric  Acid, 284 

5.  Phosphoric  Acid,                  '. 285 

6.  Solutions  of  Iodine,              285 

7.  Potassium  Hydroxide, 288 

8.  Potassium  Chlorate,             291 

9.  Potassium  Nitrate, 291 

10.  Potassium  Bichromate, 291 

11.  Sodium  Chloride, 291 

12.  Ammonia,          .         .         .         .         .         .         .         .291 

13.  Ferric  Chloride, 292 

14.  Chromic  Acid, 292 

15.  Copper  Sulphate, 293 

16.  Cuprammonia, 293 


xiv  CONTENTS. 


17.     Mercuric  Chloride,             

.     295 

18.     Millon's  Reagent,              

.     296 

296 

B.     Organic  Combinations,           . 

.     297 

20.     Alcohol,             

.     297 

21.     Ether,       .         . 

.     297 

22.     Acetic  Acid,      .         .         .         .         . 

.     297 

23.     Cupric  Acetate,         

.     298 

24.     Sodium  Nitro-Prussiate,    ..... 

.     298 

25.     Potassium  Ferrocyanide,           .... 

.     298 

26.     Oxalic  Acid,             

.     298 

27.     Asparagin,        ....... 

298 

28.     Cane  Sugar,      ....... 

.     299 

29.     Aniline  Coloring  Matter,           .... 

.     299 

30.     Aniline  Sulphate,      

.     301 

81.     Phenol,     .         .         

.     302 

32.     Phloroglucin,             

.     302 

33.     Indol,        

.     303 

34.     Eosin,       .         . 

304 

35.     Hsematoxylin,            ..... 

304 

36.     Cochineal  Extract,             

.     305 

37.     Carmine  Solutions,   

.     306 

38.    Picro-carminate  of  Ammonia, 

.     309 

39.     Alcanna  Tincture,     

.     310 

CHAPTER  V.— MICROSCOPICAL  INVESTIGATION  OF 
VEGETABLE  SUBSTANCES. 

A.     Substances  of  Universal  Distribution,         .         .         .  315 

I.  Cellulose  and  its  Modifications, 315 

1.  Cellulose  in  the  narrow  sense, 318 

2.  Mucilaginous  Cellulose, 327 

3.  Wood  Substance, 330 

4.  Middle  Lamella, 343 

5.  Corky  Cellulose, 347 

6.  Fungus  Cellulose, 353 

II.  Starch, 357 

III.  Dextrine, 365 

IV.  Vegetable  Mucilage, 367 

V.  Gum,        ....  371 


CONTENTS.  xv 

VI.  Inulin, 375 

VII.  Grape  Sugar,         .         .         . 377 

VIII.  Cane  Sugar, 378 

IX.  Albuminous  Matter, 379 

1.  Reserve  Proteid  Matter,     ......  380 

2.  Functional  Proteid  Matter, 392 

X.  Chlorophyll,      .         . 403 

XI.  Coloring  Matter  of  Flowers,             421 

XII.  Asparagin,              425 

XIII.  Inorganic  Vegetable  Elements,              ....  428 
B.     Plant  Substances  of  Limited  Distribution,         .         .431 

XIV.  Glyeoside,             432 

XV.  Tannin,           .       , 435 

XVI.  Alkaloids,              438 

XVII.  Fats, 439 

XVIII.  Essential  Oils, 441 

XIX.  Stearoptene, 441 

XX.  Resin,  Balsam,  Turpentine,             .....  442 

XXI.  Coloring  Matter  of  Flowering  Plants,  .         .         .447 

XXII.  Coloring  Matter  of  Cryptogamic  plants,                .         .  448 


CHAPTER  I. 
THE    MICKOSCOPE 


I.     INTRODUCTION. 

THE  microscope,  as  an  instrument  of  observation,  holds  at  the 
present  time  the  most  prominent  position  in  the  department 
of  scientific  botany,  especially  in  vegetable  anatomy  and  to 
some  extent  also  in  physiology.  Vegetable  anatomy  treats 
entirely  of  the  investigation  of  the  constituent  elements  of 
vegetable  organisms,  and  the  minuteness  of  these  parts  makes 
the  use  of  the  instrument  in  question  almost  always  neces- 
sary The  cultivation  of  vegetable  anatomy  has  been  so  far 
dependent  upon  the  development  of  the  microscope  that  those 
periods  in  which  most  improvements  have  been  made  in  it 
correspond  in  many  cases  to  those  in*  which  the  principal  de- 
velopment of  this  science  has  occurred.  On  the  other  hand 
also,  the  wider  development  of  phytotomy  has  not  been  without 
its  influence  upon  the  improvement  of  certain  parts  of  the  mi- 
croscope. 

The  name  of  the  apparatus  as  well  as  its  invention  belongs 
to  the  later  middle  ages,  somewhere  in  the  fifteenth  or  six- 
teenth century.  The  instrument  was  called  the  microscope 
(;j.>.7.o<>s,  small,  and  ffxo-sw,  to  look  upon,  to  observe  by  means 
of  the  sight)  because  it  allowed  very  small  objects,  grains  of 
sand,  insects  and  the  like  to  be  seen  magnified.  One  must 
not  think,  however,  that  those  first  very  timid  attempts  to 
construct  magnifying  glasses  can  in  any  way  be  compared 
with  the  instruments  fabricated  in  recent  times.  The  first  of 
these  productions  were  the  "flea-glasses,"  Vitra pulicaria  and 


THE   MICROSCOPE   IN   BOTANY. 


muscaria,  which  served  the  learned  men  of  that  time  more  as 
a  "highly  curious  and  frightful  microscopical  amusement  for 
eyes  and  mind  "  than  for  scientific  observations.  They  consisted 
of  the  kind  which  we  see  to-day  in  our  toy  shops,  and  were 
made  of  a  single  glass  lens  which  was  the  segment  of  a  sphere 
of  small  diameter.  This  lens  was  fastened  into  a  wooden  tube 
which  bore  at  its  lower  end  in  the  focus  of  the  lens  a  small  glass 
plate  on  which  a  crushed  flea,  a  gnat,  a  fly's  leg,  or  a  like  object 
was  pasted.  The  "flea-glass''  belonged  to  the  indispensable 
requisites  of  a  learned  man  of  that  time.  It  magnified  about  six 
to  ten  times,  equivalent  to  an  average  magnifying  glass  of  to- 
day. It  makes  the  most  comical  impression  on  one  to-day  to 
read  the  descriptions  which  these  Faustian  savants  give  of 
their  observations  with  the  "flea-glasses."  Many,  amazed,  de- 
scribed real  monsters  which  they  thus  observed,  while  the  less 
expert  believed  they  saw  the  Devil  himself  in  the  innocent 
instrument. 

Leeuwenhoek  (1632-1723)  one  of  the  first  who  really  set 
about  scientific  observations  with  the  microscope,  a  man  who,  by 
his  discovery  of  the  infusoria,  is  known  in  the  widest  circles, 
used  a  simple  microscope  exclusively  in  his  observations,  but 
which,  measured  by  the  ideas  of  his  time,  possessed  very  strong 
magnifying  powers.  He  understood  how  to  grind  very  perfect 
little  lenses  with  short  focal  distance,  which  he  fastened  between 
two  right-angled  plates  of  silver  or  brass  (from  4  to  5cm.  long, 
and  about  3cm.  broad)  screwed  upon  each  other  in  such  a  way 
that  one  could  look  down  through  two  corresponding  holes  in 
the  plates  and  through  the  lens  lying  between.  Behind  this 
mounted  lens  was  a  small  pointed  instrument  capable  of  move- 
ment in  all  directions,  on  the  end  of  which  the  object  to  be 
observed  was  impaled.  The  whole  instrument  wras  then  held 
towards  the  light  and  the  lens  brought  as  near  to  the  eye  as 
possible.  Leeuwenhoek's  microscopes  magnified  from  40  to  100 
times,  a  few  150,  and  one  even  270  times. 

But  already  before  the  time  of  Leeuwenhoek,  that  is,  still  be- 
fore the  close  of  the  sixteenth  century,  or  perhaps  in  the  be- 
ginning of  the  seventeenth  century,  the  compound  microscope 
had  been  invented.  It  is  very  essentially  distinguished  from 


INTRODUCTION.  3 

the  one  already  mentioned  by  this  :  that  a  picture  is  produced  by 
the  lens  nearest  the  object,  the  so-called  "objective,"  and  that 
this  image  is  viewed  through  a  lens  placed  near  the  eye,  the 
"  ocular,"  relatively  magnified.  This  principle  is  fundamental 
in  all  microscopes,  in  those  also  of  the  present  time,  though 
they  may  be  constructed  never  so  differently  from  those  of  the 
first  inventor. 

Who  should  be  regarded  as  the  inventor  of  the  first  com- 
pound microscope  was  for  a  long  time  a  doubtful  and  frequently 
disputed  question.  According  to  the  literary  studies  of  Hart- 
ing,1  it  appears  pretty  nearly  beyond  doubt  that  this  merit 
should  be  ascribed  to  two  spectacle  makers  in  Middelburg  in 
Holland,  Hans  &  Zacharias  Janssen,  father  and  son.  Pre- 
viously Fontana,  Galileo  Galilei,  and  the  Netherlander  Drebbel 
also  had  been  regarded  as  the  first  inventors  of  our  instrument. 
In  a  work  by  Borel,  a  Frenchman,  is  a  letter  from  a  countryman 
and  friend  of  the  younger  Janssen  who  among  other  things 
describes  the  first  microscope  in  the  following  way.2  "It  pos- 
sessed not,  as  such  an  instrument  would  now,  a  short  tube,  but 
one  almost  a  foot  and  a  half  long.  The  tube  itself  was  of  gilded 
brass  and  was  fixed  in  the  middle  at  a  height  of  two  fingers  on 
three  bronze-crested  dolphins.  The  foot  consisted  of  a  disk  of 
ebony  that  carried  various  small  instruments  and  minute  objects 
which  we  viewed  from  above  in  almost  miraculously  magnified 
form." 

Although  with  the  invention  of  the  compound  microscope  in 
general,  there  had  been  worked  out  the  proper  arrangement 
which  should  be  given  to  the  apparatus,  still  the  construction 
of  magnifying  glasses  during  the  century  succeeding  the  in- 
vention was  so  faulty  that  it  was  little  or  not  at  all  fitted  for 
making  those  observations  which  have  been  attained  in  recent 

O 

times. 

During  that  period  the  object  under  investigation  was  viewed 
with  reflected  light  only,  it  being  concentrated  upon  the  object 
from  the  lamp  which  was  the  source  of  light,  by  means  of  a 

1  Harting.    Das  Microskope  Braunschweig,  1859,  pp.  586-596. 

2  The  Latin  text  is  printed  in  Halting,  1.  c.,  p.  589.    The  work  of  Borel  bears  the  title 
"  De  vero  telescopii  inventore,  cum  brevi  omnium  conspiciliorum  historia.    Accedit  etiam 
centuria  observationum  microscopicai-uin.    Hag.  Comitum,  1655. 


4  THE   MICROSCOPE   IN   BOTANF. 

globe  filled  with  water,  or  by  a  condensing  lens.  Such  an  in- 
strument adapted  only  for  superficial  illumination  is  illustrated, 
for  example,  by  Robert  Hooke,3  who  also  states  that  a  thin 
section  through  a  flask  cork,  on  a  dark  background,  looks  like 
a  honey  comb,  in  which  could  be  distinguished  open  spaces 
(pores)  and  separating  walls.4  The  illuminating  reflector  (mir- 
ror) which  is  indispensable  to  us,  and  which  we  place  under  the 
object,  in  order  to  view  it  with  transmitted  light  was  first  intro- 
duced for  the  use  of  the  compound  microscope  about  the  year 
1735,  by  Culpeper  and  Scarlet,5  and  a  few  years  later  (1740) 
by  Wilson,6  for  the  simple  microscope.  Thus  an  important  step 
forward  was  made. 

But  at  that  time,  the  construction  and  combination  of  lenses 
for  the  compound  microscope  were  very  faulty.  One  did  not 
then  as  now  employ  an  ocular,  made  of  two  glasses,  and  an 
objective  of  several  lenses,  but  each  consisted  at  that  time  of  but 
one  glass.  The  consequence  was  that  the  microscopic  image 
often  appeared  very  much  bent,  because  only  its  middle  por- 
tions were  clearly  shown,  while  the  edges  were  distorted  be- 
yond recognition.  This  explains  what  Wolf7  expressly  declared, 
in  the  year  1723,  that  at  that  time  the  simple  microscope  was 
much  more  in  use  than  the  compound  (see  above,  Leeuwen- 
hoek)  and  that  one  would  much  rather  use  the  former,  es- 
pecially in  high  magnifications,  than  the  latter. 

From  the  following,  it  becomes  clear  how  these  microscopes 
gradually  gave  place  to  those  with  flat  fields  of  view.  The  in- 
struments of  Drebbel,  Galilei  and  probably  also  the  one  of 
Janssen,  above  described,  possessed,  as  remarked,  two  convex 
lenses,  the  one  serving  as  ocular,  and  the  other  as  objective. 
Fontana  inserted  in  his  microscope  an  intermediate  concave  glass 
between  the  other  two  lenses.  Hook  did  the  same,  but  princi- 
pally with  a  view  to  enlarge  the  field  of  vision,  and  he  let  it 
remain  when  he  saw  the  image  clearly  and  distinctly.  A  fur- 
ther step  was  made  towards  this  improvement  by  Divini,  about 
1670,  who  first  united  two  plano-convex  lenses  in  one  ocular, 

3  R.  Hooke,  Micrographia,  or  some  physiological  descriptions  of  minute  bodies  made 
by  magnifying  glasses.    London.    1667. 

4  J.  Sachs,  Geschichte  der  Botanik.    p.  247.        5  Harting,  1.  c.,  p.  672. 

e  Hurting,  I.  c.,  p.  615.       1  Sachs,  1.  c.,  p.  267.— See  also,  Hurting,  I.  c.,  p.  688. 


INTRODUCTION.  5 

as  it  is  done  to-day.  Soon  after  that,  doublets  also,  which 
had  already  been  in  use  for  some  time  as  simple  microscopes, 
began  to  be  employed  as  objectives.  At  last,  toward  the  end 
of  the  seventeenth  century,  there  came  in  combinations  of  lenses 
as  objectives.  They  were  either  biconvex  or  plano-convex8 
lenses  of  unlike  foci,  permitting  therefore  by  different  combina- 
tions the  production  of  lower  or  higher  magnifications,  while  in 
the  oldest  compound  microscopes  the  different  magnifications  had 
been  brought  about  in  this  way,  the  tube  was  constructed  like  that 
of  a  telescope  of  three  or  four  parts  which  slid  into  each  other, 
and  the  magnifying  power  was  increased  by  drawing  these  out. 

But  the  power  of  the  microscope  was  so  small  that  a  scholar 
of  that  time  praised  a  microscope  which  magnified  eighty  diam- 
eters, as  being  "certainly  quite  gigantic  in  its  magnification" 
(quod  certe  insigne  augmentum  est). 

AVhile  we  have  seen  that  in  the  fabrication  of  the  microscope 
it  has  gradually  come  about  that  the  spherical  aberration  has 
in  great  part  been  overcome  by  means  of  suitable  forms  and 
combinations  of  lenses,  it  was  not  until  the  concluding  third  of 
the  last  century  that  another  great  defect  of  the  instrument  was 
removed,  namely,  the  chromatic  aberration.  Xewton  had  indeed 
already,  towards  the  end  of  the  seventeenth  century,  expressed 
the  opinion  that  glasses  might  be  constructed  which  should  pro- 
duce a  colorless  image  by  a  combination  of  two  lenses,  made  of 
material  having  the  greatest  possible  likeness  of  refractive  power, 
and  the  greatest  possible  difference  in  color-dispersing  power, 
although  it  was  not  possible  for  him  to  bring  the  matter  to  ex- 
perimental proof.9  Dollond  had  indeed,  in  1758,  constructed  the 
first  achromatic  telescope,  and  so  had  reduced  that  theoretical 
calculation  to  practice.  Euler  also,  in  1771,  had  established  a 
coherent  theory  of  Achromatics,  and  Fuss  in  1774,  in  accord- 
ance with  the  analyses  of  the  latter,  gave  directions  for  con- 

8  While  it  is  naturally  impossible  to  correct  the  spherical  aberration  by  a  combination  of 
biconvex  glasses,  it  is  quite  easily  done  by  the  use  of  plano-convex  lenses,  whose  plane  side 
is  turned  under  and  brought  next  to  the  object.    Although  toward  the  end  of  the  last  cen- 
tury plano-convex  object-lenses  were  generally  used,  they  were  constantly  so  placed  that 
the  convex  side  was  turned  toward  the  object.    Chevalier  and  Amici  first  used  them  re- 
versed, and  so  reduced  the  spherical  aberration  to  a  minimum. 

9  Through  inexact  observation  he  was  led  into  theerror  of  supposingthat  materials,  with 
nearly  the  same  refractive  index  and  very  different  color-dispersing  power,  did  not  exist. 


THE   MICROSCOPE   IN   BOTANY. 

structing  an  achromatic  microscope.  But  it  was  held  to  be 
impossible  to  construct  achromatic  glasses  of  the  small  size 
required  in  microscopic  objective  lenses,  and  it  was  believed 
therefore  that  a  microscope  could  never  be  made  which  would 
give  at  the  same  time  in  some  measure  high  magnification  and  a 
clear  sharp  image. 

According  to  Hailing,10  it  was  again  a  Hollander,  van  Deyl, 
(1807),  who  first  constructed  a  really  achromatic  microscope, 
forming  his  objective  out  of  two  biconvex  crown-glass  lenses, 
and  a  biconcave  flint-glass  lens  lying  between.  His  instrument, 
however,  for  reasons  stated  in  note  8,  p.  5,  possessed  a  very 
considerable  spherical  aberration. 

A  short  time  after  that,  Brewster  made  the  attempt  to  substi- 
tute a  fluid  for  the  flint-glass  middle  lens  (Newton  had  attempted 
to  do  the  same  thing  already).  It  is  known  under  the  name  of 
Brewster's  achromatic  globe.  It  is  a  glass  globe  filled  with  water 
in  which  two  biconvex  lenses  are  made  to  lie  pole  to  pole  in  the 
optical  axis.  The  contrivance  has  however  never  come  into  use. 

After  achromatic  objective  lenses  for  microscopes  had  been 
prepared  by  Fraunhofer,  the  most  gifted  optician  of  all  time, 
there  appeared  in  France,  Chevalier  (about  1824),  and  in  Italy, 
Arnici  (1827  and  later),  who  constructed  objectives  such  as 
to-day  are  generally  in  use,  under  the  designation  of  "apla- 
natic."]  In  them  the  spherical,  as  well  as  the  chromatic 
aberration,  was  so  far  suppressed  that  they  no  longer  essentially 
hindered  microscopical  observation.  We  shall  become  more 
intimately  acquainted  with  the  aplanatic  lens  in  the  subsequent 
portions  of  this  chapter. 

The  names  which  in  more  recent  times  have  had  most  signif- 
icance in  microscope-making,  and  with  which,  together  with  their 
contributions  we  shall  become  more  intimately  acquainted,  in  the 
proper  place  hereafter,  are  principally,  Hugo  von  Mohl,  Ober- 
hauser,  Hartnack,  Nachet,  Merz,  Plossl,  Beneche,  Wasserlein, 
Pritchard,  Ross,  Zeiss,  Seibert,  Krafft,  Winkel,  Schieck,  Leitz, 
Powell  and  others.* 

10  Hartins,  L  c.,  p  691. 

11  From  d  privative  and  n-Aacaw,  to  deceive,  to  lead  astray. 

*  Certainly  the  names  of  our  two  greatest  American  opticians,  Spencer  and  Tolles,  should 
not  be  omitted  from  this  list.  A.  B.  H. 


INTRODUCTION.  7 

In  our  historical  review,  we  have  thus  far  drawn  attention 
only  to  the  optical  part  of  the  microscope,  while  the  bearer  of 
these  parts,  the  stand,  has  been  left  out  of  sight. 

In  the  period  directly  after  the  invention  of  the  instrument, 
very  little  attention  was  devoted  to  the  microscope  stand.  It 
commonly  consisted  of  an  elaborately  turned  piece  of  wood, 
while  the  cylindrical  draw-tube  was  often  made  from  pasteboard. 
The  first  important  improvement  which  the  mounted  compound 
microscope  received  was  the  introduction  of  the  illuminating 
mirror  by  Culpeper  and  Scarlet,  as  already  noticed  above.  The 
inicroscopist  now  began  to  work  with  transmitted  light.  The  ob- 
ject for  observation  was  placed  upon  a  perforated  plate,  fche  stage, 
which  occupied  a  position  between  the  mirror  and  the  objective. 

In  order  to  focus  the  object,  that  is  to  say,  to  bring  it  ex- 
actly in  the  focal  point  of  the  object-glass,  we  may  proceed  in 
either  of  two  ways,  viz.,  the  object  table  or  stage  may  be  made 
fast,  and  the  adjustment  produced  by  moving  the  tube  which 
carries  the  optical  apparatus  towards  it,  or  on  the  other  hand 
the  tube  may  be  fixed  and  a  vertical  movement  may  be  given  to 
the  stage.  In  the  first  compound  microscopes  with  illuminating 
mirrors,  from  the  factory  of  Culpeper  and  Scarlet,  the  stage 
was  fixed,  and  the  focussing  was  done  by  shoving  the  tube  by 
hand.  But  already,  about  the  middle  of  the  eighteenth  century, 
Cuff  had  employed  the  setting  screw  to  accomplish  a  more  exact 
focussing.  In  his  microscopes  the  tube  was  fastened  to  a  metal 
hinge  which  could  move  up  and  down  on  a  perpendicular  metal 
rod.  By  this  manipulation,  the  approximate,  or  so-called ,  coarse 
adjustment  was  accomplished,  while  by  a  clamping  screw,  the 
hinge  was  made  fast,  and  a  very  small  shortening  or  lengthening 
of  the  hinge  itself  was  accomplished  by  a  second  screw  (the  so- 
called  micrometer  screw)  thereby  making  the  fine  adjustment. 
In  later  microscopes  (of  Martin,  Jones,  van  Deyl,  etc.)  the 
tube  was  moved  by  a  simple  rack  and  pinion. 

In  the  instruments  of  Chevalier  the  opposite  plan  was  adopted, 
the  stage  was  moved  by  means  of  a  hinge, -*vith  a  clamp  screw 
on  a  vertical  prismatic  metal  rod.  The  fine  adjustment  was 
produced,  as  in  the  instruments  of  Jones,  by  means  of  a  care- 
fully cut,  small  thread,  fine-adjustment  screw. 


THE   MICROSCOPE   IN   BOTANY. 

Afterwards  these  last  contrivances  were  generally  abandoned  ; 
only  the  microscopes  of  Amici  now  have  them,  and  in  the  oldest 
stands  of  Plossl  both  the  tube  and  the  stage  are  movable  on  the 
same  vertical  rod. 

In  the  instruments  of  the  present  time  (or  at  least  on 
the  larger  and  medium  stands),  without  exception,  the  adjust- 
ment is  produced  by  moving  the  tube  vertically  in  a  direction 
perpendicular  to  the  stage,  which  is  made  fast  to  the  rod.  The 
coarse  adjustment  is  produced  directly  by  free  hand  or  by 
means  of  a  rack  and  pinion.  The  fine  adjustment  is  made  by 
a  fine  screw. 

While  .the  stands  of  the  first  microscopes,  which  were  made 
throughout  of  polished  or  unpolished  wood,  could  satisfy  but 
very  modest  demands  as  to  their  outward  appearance,  stands 
made  of  brass  came  into  very  general  use  at  the  end  of  the  last 
century.  There  came  a  time  when  the  outside  appearance  of 
the  stand  received  by  far  the  most  attention,  while  the  optical 
part  was  very  little  improved.  There  was  brought  into  use 
with  the  stand  also,  at  that  time,  every  possible  useless  acces- 
sory (the  so-called  microscopical  accessory  apparatus)  so  that 
the  observer  was  often  more  hindered  than  helped  by  it  in  his 
work.12 

It  was  the  famous  phytotomist  Hugo  von  Mohl  who  first  ex- 
pressly demanded  the  simplest  constructed  microscopes,  and 
rebuked  with  sharp  words  the  coquettish  toying  with  stands 
which  was  becoming  the  fashion.  He  speaks  about  it,13  for  ex- 
ample, as  follows.  "The  simpler  the  construction  of  the  micro- 
scope is,  the  more  easily  and  more  quickly  will  one  accomplish 
all  the  necessary  movements.  The  more  complicated  the  con- 
struction the  more  will  they  cost  in  time  and  reflection,  and  the 
more  will  the  attention  be  distracted  thereby  during  the  observa- 
tion. Whoever  has  not  the  manual  dexterity  to  work  with  a  sim- 
ply constructed  microscope,  and  finds  it  necessary  to  use  a  screw 


12  The  author  has  recently  had  :m  opportunity  to  take  a  look  at  snch  an  English  instru- 
ment in  the.  physical  collection  of  the  technical  high  school  in  Brunswick,  of  which  one 
literally  cannot  get  at  the  stage  on  account  of  screws,  magnif3-ing  glasses  and  other  things, 
and  which  one  can  recognize  as  a  microscope    geneially  only  after   the  most    exact 
consideration. 

13  H.  v.  Mohl,  Mikrographie,  Tubingen,  1846,  p.  89. 


INTRODUCTION. 


instead  of  his  fingers  for  every  movement,  is  on  that  account 
disqualified  for  a  microscopical  observer,  for  he  will  labor  in 
vain  to  prepare  a  usable  specimen." 


After  this  short  historical  survey  of  the  invention  of  the  mi- 
croscope, we  will  now  turn  to  a  consideration  of  the  instrument 
itself.  Since  we  shall  assume  that  those  general  laws  of  optics 
concerning  the  refraction  of  rays  of  light,  which  may  be  found 
stated  in  every  text-book  are  already  known,  it  will  be  our  aim 
in  the  following  treatise  to  give  a  representation  of  the  microscope 
without  admitting  special  theoretical  deductions.  Afterwards 
the  reader  will  be  made  acquainted  with  the  methods  of  prepar- 
ing botanical  microscopic  specimens,  and  at  last  there  will  be 
shown  to  him  how  their  methodical  investigation  should  be 
conducted. 

But  it  will  not  perhaps  be  useless  to  insert  here  first  of  all  the 
following  general  remarks. 

From  the  start  it  must  be  clearly  understood  that  the  micro- 
scope is  not  an  instrument  to  which  one  only  needs  to  turn  and 
look  in,  in  order  to  behold  some  great  discovery.  The  micro- 
scope is,  on  the  contrary,  an  instrument  whose  use  and  manage- 
ment must  be  learned,  but  which  then,  if  it  be  used  with 
understanding  and  with  regard  for  the  most  careful  precautions, 
permits  things  to  be  seen  which  would  be  forever  shut  out 
from  the  unaided  eye. 

Under  the  microscope,  however,  we  see  always  only  a  very 
small  part  of  a  natural  body,  and  what  is  more  important,  we 
see  that  only  in  two  dimensions,  namely,  in  length  and  breadth. 
We  can  never  at  the  same  time  perceive  its  thickness.  We 
must  therefore,  in  order  to  come  to  a  clear  conception  of  the 
microscopical  structure  of  organs  having  a  corporeal  appearance, 
to  the  naked  eye,  contemplate  different  sections  through  it, 
made  in  the  direction  of  the  three  dimensions  of  space,  and  then 
combine  these  by  means  of  our  mental  eye.  Thus  there  is  re- 
quired for  seeing  and  understanding  the  microscopic  image,  not 
only  activity  of  sense  but  activity  of  mind,  also.  I  quote  here, 
ill  reference  to  this,  the  declaration  of  one  of  the  most  highly 


10  THE   MICROSCOPE   IN   BOTANY. 

accomplished  of  living  observers,  Julius  Sachs,  as  he  writes 
in  the  History  of  Botany  :M  "  Seeing  is  an  art  which  must  be 
learned  and  cultivated,  a  definite  purpose  must  stimulate  the  will 
of  the  observer,  to  will  to  see  exactly  and  rightly,  to  distin- 
guish and  combine  what  is  seen."  ....  "By  the  invention  of 
the  microscope  the  eye  became  capable  not  merely  of  seeing 
small  things  larger,  and  in  general  of  seeing  the  invisibly  small, 
but  much  more  ;  there  was  combined  with  the  use  of  magnifying 
glasses  the  one  other  advantage,  viz.  :  that  then  first  we  learned 
in  general  to  see  exactly  and  scientifically.  In  that  we  armed 
the  eye  with  a  magnifying  glass,  the  attention  was  concentrated 
on  a  single  point  of  the  object,  the  seeing  was  in  part  indistinct 
and  always  of  but  a  small  part  of  the  whole  object.  The  per- 
ception of  the  visual  nerve  must  be  accompanied  with  a  purpose- 
ful and  intense  reflection,  in  order  to  make  the  object  observed 
in  fragments  by  the  magnifying  glass,  clear,  in  its  inner  connec- 
tions, to  the  mental  eye.  So  the  eye,  by  being  armed  with  the 
microscope,  became  itself  a  scientific  instrument,  which  no 
longer  ran  over  the  objects  with  thoughtless  movements,  but 
received  strict  discipline  from  the  understanding  of  the  observer, 

and  was  kept  to  methodical  work." "As  in  every 

other  science,  so  in  the  investigation  of  the  structure  of  plants, 
the  sense  perception  must  be  worked  over  by  the  understanding, 
to  distinguish  the  important  from  the  unimportant,  and  to  bring 
the  single  perceptions  into  logical  coherence,  to  follow  a  purpose 
in  the  investigation,  but  this  purpose  can  be  none  other  in  the  last 
instance  for  the  plant  anatomist,  than  that  the  whole  inner 
structure  of  the  plant  in  its  collective  coherency  shall  be  so 
clearly  comprehended  that  it  may  at  any  time,  in  all  its  details, 
be  perfectly  reproduced  in  perfectly  sensible  definiteness  from 
the  imagination.  To  attain  this  is  not  easy,  because  the  more 
powerfully  the  microscope  magnifies  the  smaller  the  part  of  the 
whole  which  it  shows.  Skilful  and  superior  preparations,  care- 
ful combinations  of  different  images,  and  long  practice  are 
necessary  to  attain  that  object.  The  history  of  vegetable  anat- 
omy shows  how  difficult  it  has  been  for  observers  to  gradually 
form  a  clear,  coherent  conception  from  these  fragmentary  views  J' 

"  Sachs,  1.  c.,  p.  236,  ff. 


INTRODUCTION.  11 

But  there  is  also,  for  those  who  have  already  attained  some 
facility  in  the  use  of  the  microscope,  one  important  aid  which 
is  indispensable  to  methodical  observation.  Every  one  knows, 
and  has  often  been  compelled  to  make  the  remark  to  himself, 
that  our  memory  can  be  said  to  be  trustworthy  only  to  a  limited 
extent,  and  that  the  subjective  impression  which  the  brain  re- 
ceives is  but  imperfectly  and  often  but  temporarily  fixed.  It 
is  especially  necessary  to  resort  to  the  help  of  the  graphic  art 
if  one  would  preserve  in  the  memory,  with  any  exactness,  a 
series  of  microscopical  observations.  This  may  be  done  in 
either  of  two  ways.  In  the  first  place  by  keeping  an  exact 
record  of  the  observations,  and  in  the  second  place  by  endeavor- 
ing to  fix  the  microscopic  image  on  paper  by  means  of  pencil 
or  brush,  in  other  words  by  drawing  it.  The  latter  presupposes 
a  real  manual  dexterity,  a  certain  technique.  But  it  will  not 
be  difficult  for  microscopists  who  are  but  little  practised  in  draw- 
ing to  acquire  the  necessary  skill  for  this  purpose.  Besides 
this  there  is,  as  we  shall  show  farther  on,  abundant  and  man- 
ifold apparatus,  which  allows  the  microscopic  image  to  be 
thrown  down  by  means  of  a  reflecting  prism  upon  a  sheet  of 
paper  tying  near  the  microscope,  where  it  may  be  traced  with 
the  pencil  without  further  trouble.  But  these  contrivances  are 
to  be  used,  especially  by  the  beginner,  only  with  the  greatest 
care.  For  the  microscopical  drawing  should  not  be  merely  the 
spiritless  crude  copy  of  the  image  seen,  but  it  should  receive 
into  itself  all  the  experiments,  all  the  studies  which  the  observer 
has  made  upon  the  object ;  in  a  word  it  should  be  idealized.  In 
connection  with  the  opinion  just  now  expressed  I  will  quote  a 
restrictive  remark  of  Sachs:  "A  microscopical  drawing,  like 
illustrations  of  objects  of  natural  science  generally,  cannot  quite 
lay  claim  to  replace  the  object  itself;  all  the  more  then  should 
it  repeat  with  all  distinctness  what  the  observer  has  perceived 
and  in  so  far  support  the  description  in  the  text.  The  drawing 
will  be  all  the  more  perfect,  the  more  the  eye  is  trained  in  ob- 
serving and  the  understanding  in  interpreting  the  forms.  The 
illustrations  should  show  to  the  reader  nothing  other  than  what 
has  been  traversed  throughout  by  the  mind  of  the  observer, 
for  only  so  can  it  serve  to  bring  the  two  to  a  mutual  understand- 


12  THE   MICROSCOPE   IN  BOTANY. 

ing.  But  the  case  has  still  another  significance :  even  during 
the  drawing  of  a  microscopic  object  it  is  necessary  for  the 
eye  to  dwell  on  single  lines  and  points,  in  order  to  comprehend 
their  true  dependence  in  respect  to  all  the  dimensions  of  space  ; 
it  often  happens  thence  that  relations  are  perceived,  which,  pre- 
viously, even  in  the  most  careful  observation,  were  not  noticed  ; 
for,  however  the  question  being  investigated  is  determined,  new 
questions  are  opened.  Thus  as  the  eye  is  first,  by  the  use 
of  the  microscope,  trained  to  scientific  seeing,  so  first  by  careful 
drawing  of  the  object  will  the  educated  eye  become  a  growing 
councillor  of  the  investigating  mind."15 

One  already  skilled  in  drawing,  when  he  begins  to  make  mi- 
croscopical observations  and  drawings,  will  at  first  produce 
quite  imperfect  pictorial  representations ;  but  by  the  constant 
use  and  practice  of  the  eye  in  microscopical  seeing,  the  drawings 
will  tend  to  ever  greater  perfection  and  instructiveness. 

Much  has  been  written  as  to  the  personal  characteristics  which 
the  microscopist  should  possess.  We  limit  ourselves,  therefore, 
under  the  supposition  of  making  a  theoretical  presentation,  to 
the  four  following  factors  required  by  him  :  a  skilful  hand, 
good  eyes,  a  tranquil  mind,  and  self  knowledge. 

For  the  preparation  of  microscopical  specimens,  which  pre- 
supposes the  careful  management  of  the  most  various  tools,  a 
certain  skilfulness  of  hands  is  one  of  the  first  conditions.  In 
relation  to  the  eyes,  the  short-sightedness  so  common  to  scien- 
tifically educated  people,  is  in  no  way  a  hindrance  to  microscop- 
ical observation.  On  the  contrary,  it  is  often  very  useful  in  the 
preparation  of  specimens,  as  a  brilliant  example  may  be  quoted 
to  show,  in  the  case  of  one  of  the  most  accomplished  micros- 
copists  and  vegetable  anatomists,  Wilhelm  Hofmeister,  who,  in 
preparing  objects,  brings  them  very  close  before  his  imspecta- 
cled,  Very  shortsighted  eyes,  and  in  this  way  sees  relatively 
very  large,  thus  using  the  eyes  in  place  of  a  mounting  micros- 
cope.16 Observe  with  the  right  eye,  but  one  should  avoid 

16  Sachs,  1.  c.,  p.  280. 

16  Spectacle  wearers  do  best  when  they  remove  the  glasses  during  exact  microscopical 
observation,  and  they  should  be  used  in  preparing  microscopical  drawings,  only  by  ex- 
tremely shortsighted  people,  when  it  cannot  be  done  without  them.  In  this  manipulation 
there  frequently  results  (at  least  to  the  shortsighted  author)  this  discomfort :  since  the  right 


INTRODUCTION.  13 

pinching  up  the  left  eye  meanwhile,  because  the  muscles  used 
in  shutting  it  will  soon  thereby  become  sensibly  affected.  One 
should  look  at  the  same  time  with  the  left  eye  upon  the  table 
(the  darkest  possible,  for  example,  a  dark  green  colored  one). 
The  inclination  to  squinting,  arising  from  this  use  of  the  eyes, 
may  be  easily  prevented  by  giving  the  eyes  timely  rest  after 
working  with  the  microscope.* 

Respecting  the  mental  condition  of  the  observer  it  may  be 
mentioned,  that  —  as  it  lies  in  the  nature  of  the  case  —  only 
that  temperament  of  mind  can  be  serviceable  to  investiga- 
tion in  which  we  find  ourselves  absolutely  passionless.  Says 
Harting,17  "  But  for  the  exercise  of  the  critical  judgment,  in 
microscopical  observations,  there  is  need  not  merely  of  the  sim- 
ple purpose,  but  we  must  find  ourselves  in  that  condition  of 
mind  which  makes  it  possible  for  us  to  see  with  unclouded 
vision,  and  to  judge  with  unprejudiced  understanding.  As  the 
principal  requirement  thereto  I  name  mental  tranquillity  during 
the  investigation.  As  easy  as  it  might  seem  to  satisfy  this 
requirement,  experience  teaches  that  the  opposite  most  often 
prevails.  In  microscopical  investigations  this  is  of  great  im- 
portance ;  for  these  often  occasion  lively  mental  impressions 
which  are  incompatible  with  the  desired  mental  rest  during  the 
observations." 

Of  the  four  above  named  indispensable  characteristics  of  the 
microscopist,  self  knowledge  plays  the  chief  role.  The  micros- 
copist  must  be  a  sceptic  through  and  through ;  he  must  take 
nothing  for  granted  ;  he  must  approach  every  object  to  be  inves- 
tigated with  a  certain  mistrust.  He  will  much  more  easily  be 
trained  to  be  a  good  observer  if  he  continually  seek  to  detect 
himself  in  a  false  observation,  than  if  he  goes  at  his  work  with 
the  self-satisfied  consciousness  that  nothing  could  make  him  see 

spectacle  glass  so  constantly  strikes  against  the  ocular,  the  part  which  lies  upon  the  nose 
will  be  pressed  upon  the  skin  covering  the  bridge  of  the  nose,  in  such  a  way  as  finally  to 
produce  a  very  troublesome  pain,  which  may  bring  on  headache.  This  discomfort  may  be 
avoided  by  soldering  upon  the  bearing  place  of  the  spectacles  a  thin  plate  of  gold  some 
three  millimeters  wide,  and  35  to  40  mm.  long,  which  has  previously  been  bent  into  the  ex- 
act form  of  a  nose  saddle. 

17  Harting,  I.  c.,  page  327. 

*  The  evils  here  mentioned  are  greatly  mitigated  by  the  use  of  the  binocular  microscope, 
or.  with  monocular  instruments,  by  employing  an  eye  shade  such  aa  that  shown  in  Fig.  17. 
B.H.W. 


14  THE   MICROSCOPE   IN   BOTANY. 

anything  falsely  or  imperfectly.  The  microscopist  must  exer- 
cise self-criticism.  As  easy  as  it  is  to  criticise  others,  it  is 
very  difficult  for  many  men  —  perhaps  for  all  —  to  lay  upon 
themselves  the  strict,  critical  measuring  rod,  with  which  they 
are  so  ready  to  measure  others.  Egoism,  plainly,  rules  the 
world.  Yea,  still  more,  not  only  is  self-criticism  necessary  to 
the  microscopist,  but  also  love  of  truth  for  its  own  sake.  He 
should  not  delude  himself,  by  thinking  that  he  has  already  seen 
this  or  that  with  exactness,  or  perfect  correctness,  when  it  has 
first  darkly  dawned  upon  his  consciousness.  He  must  con- 
stantly protect  himself  from  accepting  for  positive  fact,  what  is 
but  probability  or  only  possibility.  How  this  last-named  qual- 
ity of  the  microscopist,  and  of  all  who  would  become  such,  is 
to  be  attained  is  not  easily  put  down  in  words  on  paper,  —  in 
respect  to  that  every  one  must  go  through  his  own  self-discipli- 
nary school, —  but  the  inscription  upon  the  Delphian  temple, 
"Know  thyself,"  should  in  spirit  constantly  hover  before  him. 

II.     THE  COMPOUND  MICROSCOPE. 

The  compound  diopteric  microscope18  is  an  optical  apparatus 
which,  by  means  of  a  convex  lens,  produces  a  magnified  image 
of  an  object,  which  again  is  viewed  through  a  glass  that  still 
further  enlarges  it.  The  compound  is  distinguished  from  the 
simple  microscope  essentially  by  this  fact,  that  by  means  of  the 
latter  we  view  the  magnified  object  itself,  while  by  the  former 
we  look  only  upon  the  enlarged  image  of  the  object.  From 
this  statement  it  must  follow  that  the  compound  microscope 
must  consist  of  at  least  two  glasses,  viz.,  of  one  which  is 
brought  near  to  the  object  to  be  magnified,  and  which  produces 
the  image  (image  producer,  objective),  and  of  a  second  which 
stands  near  to  the  observing  eye,  and  through  which  the  picture 
already  produced  is  viewed  (image  viewer,  ocular).  The  two 
glasses  must  naturally  have  such  a  mutual  position  in  respect  to 
each  other  that  the  image  will  Ml  exactly  in  the  focus  of  the 
ocular.  In  microscopes  of  the  present  day,  however,  neither 

"Compound  catopteric  microscope  there  is  none  — the  catadiopteric  is  temporarily  ex- 
cluded from  our  consideration. 


PL.    III. 


Zentmayer's  American  Student  Stand. 


THE   MICROSCOPE   STAND.  15 

the  objective  nor  the  ocular  consists  of  only  one  lens,  but  the 
former  is  [most  commonly]  constructed  of  three  plano-convex 
achromatic  lenses,  and  the  latter  of  two  glasses  of  which  the 
one  (  the  collecting  lens  )  is  placed  beneath  the  image  and 
[sometimes]  has  a  biconvex  form,  while  the  true  ocular  is  above 
the  image  and  is  plano-convex  with  the  plane  side  turned 
toward  the  eye.  For  the  production  of  a  good  microscopic 
image  it  is  required  : 

#.  That  these  lenses  shall  by  skilful  grinding  be  given  that 
curvature  which  corresponds  to  the  magnification  as  it  has  been 
previously  determined  by  calculation. 

b.  That  the  five  lenses  shall  be  exactly  centered  ;  that  is,  that 
their  foci  shall  lie  in  one  straight  line  in  the  longer  axis  of  the 
microscope. 

The  above  described  optical  apparatus  must  be  mounted  in 
a  stand  so  constructed  as  to  permit : 

a.  Objective  and  ocular  to  be  placed  at  a  definite  distance 
from  each  other  which  remains  unchanged  during  the  observation. 

b.  The  bringing  of  the  object  to  be  observed  exactly  in  the 
focus  of  the  objective. 

c.  The  furnishing  a  sufficiently  large  quantity  of  light  for  the 
object,  to  give  its  image  the  desired  degree  of  brightness. 


I.     THE  MICROSCOPE   STAND. 

[For  the  purpose  of  introducing  the  forms,  modifications  and 
nomenclature  of  the  various  parts  of  the  compound  microscope 
as  now  made  in  this  country,  a  few  stands  will  be  figured  and 
described  ;  the  selection  being  made  with  a  view  to  obtain  (with 
the  exception  of  plates  VII  and  VIII)  typical  American  forms, 
of  moderate  size  and  free  from  ostentatious  display  of  unnec- 
essary mechanism,  and  especially  those  which  have  been  instru- 
mental in  bringing  recent  improvements  into  use.] 

[Stands  of  the  class  represented  by  plates  III  to  VI,  varying 
in  style  according  to  the  skill,  ingenuity,  or  caprice  of  their 
makers,  can  be  obtained  from  all  dealers  at  a  cost  of  from  $25 
to  830,  or  with  a  minimum  outfit  of  objectives  and  accessories 
at  $40  to  $50.  Stands  of  the  class  represented  by  plates  IX, 


16  THE   MICROSCOPE   IN   BOTANY. 

X  and  XI,  should  cost  with  a  minimum  outfit  about  $50 
to  $75  ;  though  they  are  capable,  by  a  judicious  increase  of  ex- 
penditure, according  to  the  needs  or  means  of  the  purchaser, 
of  being  developed  into  instruments  of  a  far  higher  and  costlier 
grade.] 

[A.    THE  STUDENT  MICROSCOPE.] 

[As  a  sample  of  the  smallest,  simplest,  and  least  expensive 
instruments  really  worthy  of  being  commended  as  available  for 
scientific  work,  may  be  mentioned  the  ?f  Student "  microscope  of 
Joseph  Zentmayer  of  Philadelphia,  which  is  represented  at  one- 
third  actual  size  in  plate  III ;  the  plate  representing  the  parts 
usually  known  in  the  aggregate  as  the  stand.] 

[The  foot  is  a  support  widely  spread  at  the  bottom,  having 
three  points  of  rest  upon  the  table,  and  prolonged  upward  at 
the  center  into  a  conical  pillar,  which  bears  at  its  summit,  by  a 
trunnion  joint  cnpable  of  rest  in  any  position  from  vertical  to 
horizontal,  the  parts  required  for  holding,  illuminating  and 
viewing  the  object.] 

[The  stage,  occupying  a  central  position,  is  a  firm  plate  of 
blackened  brass,  nearly  square  in  form,  perforated  in  the  optical 
axis  of  the  instrument  with  a  circular  opening  for  the  transmis- 
sion of  light,  and  designed  to  support  the  object.  A  glass  slip, 
or  other  contrivance  carrying  the  object,  is  held  in  position, 
while  lying  upon  the  stage,  by  two  spring  clips  under  which  it 
is  placed.  The  size  of  the  central  opening  of  the  stage  and 
the  amount  of  light  passing  through  it,  are  regulated  by  means 
of  a  circular  revolving  plate  or  diaphragm,  let  into 'its  upper 
surface,  and  supplied  with  a  series  of  apertures  of  various  sizes, 
any  one  of  which  may  easily  be  brought  into  use.] 

[The  illuminating  portion  is  a  mirror,  plane  on  one  side  and 
concave  on  the  other,  placed  below  the  stage,  and  so  mounted 
that  it  can  be  readily  turned  toward  any  source  of  light.  It 
is  supported  by  a  tail-piece  or  mirror-bar,  a  radial  arm  having 
a  swinging  motion  around  a  center  corresponding  with  the  po- 
sition of  the  object  on  the  stage,  by  which  motion  any  desired 
obliquity  of  light  can  be  obtained  with  great  facility,  or  the 


PL.  IV. 


The   Model    Microscope 


THE   MODEL   MICROSCOPE.  17 

• 

mirror   can    be  carried  above  the  plane  of  the  stage  for  the 
purpose  of  reflecting  light  upon  the  top  of  opaque  objects.] 

[The  main  tube  of  the  instrument  is  called  the  compound 
body.  It  contains  an  ocular,  or  eye-piece,  slipped  into  its  upper 
end  and  has  a  screw  at  its  lower  end  for  the  reception  of  any 
desired  objective.  Its  normal  length  for  use  in  an  inclined 
position,  as  shown  in  the  plate,  is  partly  secured  by  means  of 
an  inside  sliding  tube,  known  as  the  draw-tube,  which  can  be 
pushed  in  for  the  sake  of  greater  compactness  when  the  stand 
is  to  be  used,  as  is  frequently  necessary  in  laboratory  work,  in  a 
vertical  position.  The  whole  compound  body,  carrying  the 
essential  optical  parts  of  the  microscope,  slides  smoothly  through 
a  fixed  outside  tube,  so  that  the  required  distance  between  the 
magnifiers  and  the  object  can  be  approximately  secured  by  a 
push  witli  the  hand.  By  using  the  thumb  and  fingers  adroitly, 
and  giving  a  screwing  motion  to  the  sliding  tube,  this  adjust- 
ment can  be  made  safely,  and  with  sufficient  precision  for  even 
moderately  high  powers.  When  greater  accuracy  is  required 
it  is  attained  by  means  of  the  fine  adjustment,  a  sliding  motion 
upon  planed  surfaces  of  brass  just  back  of  the  compound  body, 
this  motion  being  controlled,  with  great  delicacy,  by  means  of  a 
screw  with  finely  cut  threads  acting  upon  an  intervening  lever. 
The  milled  head  attached  to  the  top  of  this  screw  appears  on 
the  extreme  left  of  the  instrument,  at  the  top  of  the  curved 
limb  connecting  the  stage  with  the  compound  body.] 

[33.     THE  MODEL  MICROSCOPE.] 

[This  microscope,  made  by  the  Bausch  and  Lonib  Optical 
Co.,  of  Kochester,  N.  Y.,  is  represented  in  plate  IV,  one-third 
natural  size.  It  is  a  rather  larger  instrument,  of  convenient 
form  and  good  workmanship,  having  two  pillars  instead  of  one. 
The  coarse  adjustment  is  made  by  a  rack  and  pinion  movement, 
the  large  milled  heads  of  the  pinion  appearing  in  the  plate  just 
behind  the  compound  body ;  this  adjustment  being  more  con- 
venient though  not  more  precise,  in  skillful  hands,  than  the 
adjustment  by  sliding  tube.  The  fine  adjustment  screw  is  in 
the  same  position  as  before,  though  acting  upon  a  clock-spring 
2 


18  THE  MICROSCOPE   IN   BOTANY. 

system,  to  be  described  hereafter,  instead  of  upon  a  lever. 
The  stage  is  round,  and  concentric  to  the  optical  axis  of  the 
instrument,  as  are  all  other  round  stages  worth  mentioning ; 
and  to  it  may  be  added  an  extra  revolving  plate  with  a  movable 
object-carrier,  by  which  means  the  adjustment  of  the  object 
beneath  the  objective  is  much  facilitated.  The  diaphragm,  seen 
just  beneath  the  stage,  or  other  substage  apparatus,  is  slipped 
into  a  substage  ring  provided  for  that  purpose.] 

[C.     THE  ACME  MICROSCOPE]. 

[Somewhat  similar  to  the  last  in  size  and  general  efficiency  is 
the  new  model  Acme  No.  4,  represented  in  Plate  Y,  made  and 
sold  by  James  W.  Queen  &  Co.,  of  Philadelphia.  In  this  in- 
strument the  fine  adjustment  screw  is  removed  to  an  exceptional 
location  below  the  limb,  and  the  pinion  of  the  coarse  adjustment 
is  placed  very  high,  close  to  the  top  of  the  limb,  in  order  to 
secure  the  long  range  of  adjustment  required  for  low-power 
objectives.  The  diaphragm,  being  attached  to  a  movable  arm, 
can  be  swung  out  of  position,  as  seen  in  the  cut,  when  not  in 
use,  and  a  substage  ring,  also  shown  in  the  cut,  attached  in  its 
place  for  the  reception  of  an  illuminating  lens  or  other  ap- 
paratus. A  material  advantage  of  this  stand  is  the  possession 
of  a  body  sufficiently  large  for  oculars  of  ample  size ;  thus 
admitting  adequate  oculars  of  its  own  and  permitting  the  fre- 
quently convenient  interchange  of  any  oculars,  not  exceeding 
that  reasonable  size.  The  diameter  of  the  ocular  is  about  1 J  inch 
(32  mm.),  which  is  the  size  recently  recommended  as  a  standard 
by  the  committee  on  oculars  of  the  American  Society  of 
Microscopists,  but  not  yet  acted  upon  by  the  society.  A 
larger  and  more  elaborate  Acme  stand,  No.  3,  by  the  same 
manufacturers,  has  a  body  of  the  same  size,  but  possesses  a 
rotating  stage  and  a  substage,  somewhat  like  those  in  Plate  X ; 
the  substage  being  attached,  however,  to  the  same  bar  as  the 
mirror,  as  in  Plate  IX.] 

[D.     THE  NEW  STUDENT  STAND.] 

[Another  excellent  instrument  of  the  same  grade,  of  the 
latest  American  type,  is  the  New  Student  Stand,  made  by 


PL.   V. 


The  Acme   Microscope. 


PL.  VI. 


Bulloch's   New  Student  Stand. 


THE   PHYSICIANS'   STAND.  19 

Walter  II.  Bui  loch  of  Chicago,  and  represented  f  natural  size 
in  Plate  VI.  The  peculiarity  of  this  instrument,  as  compared 
with  the  preceding,  is  the  possession  of  Mr.  Bulloch's  form  of 
fine  adjustment  described  hereafter.  A  sliding  object-carrier 
which  can  be  adapted  to  the  stage  is  shown  lying  near  the  foot 
of  the  stand.] 

[E.     THE  PHYSICIANS'  STAND.] 

A  very  solid  and  serviceable  instrument  of  this  type  is  the 
Physicians'  Microscope  of  L.  Schrauer  of  New  York,  shown 
in  Plate  VIII  A.  The  body  is  large,  admitting  an  ocular  of 
32  mm.  in  diameter,  and  is  adjustable  by  means  of  its  draw-tube 
to  any  length  from  16  to  25  cm.  or  more.  The  diaphragm  is 
inserted  in  the  stage ;  and  a  glass  sliding  stage  is  provided,  in 
the  Zentmayer  style,  held  in  position  by  a  spring  with  ivory 
tip.  Such  a  stage  has  a  smooth  motion  and  wide  range,  is 
available  for  use  Avith  the  Maltwood  finder  (a  photographed 
scale  of  great  use  for  recording  the  exact  location  of  mounted 
objects  on  a  slide  and  enabling  them  to  be  promptly  found  when 
wanted  again),  and  is  unaffected  by  those  reagents  which  might, 
in  certain  cases,  mar  a  brass  stage.  The  joint  by  which  this  stand 
is  inclined  has  a  set-screw  for  securing  it  in  any  position.  The 
disk  of  the  swinging  mirror-bar  is  graduated  as  in  all  the  higher 
class  stands  of  this  type,  for  the  purpose  of  determining  the 
obliquity  of  illumination  or  the  angular  aperture  of  objectives.] 

[P.     THE   (CONTINENTAL)   STUDENT  STAND.] 

[\Vhile  the  stands  heretofore  and  hereafter  described  may  be 
considered  as  representing  the  characteristic  American  type, 
there  have  always  been  some  observers  who  preferred  the  small, 
compact  stands  of  the  French  and  German  model,  known  as  the 
"  continental "  style.  Some  makers  have  accordingly  adopted  a 
model  of  this  type.  Mr.  J.  Grunow,  of  Xew  York,  one  of  the 
earliest  American  makers,  has  always  been  distinguished  for  this 
class  of  stands,  and  his  excellent  workmanship  has  gone  far 
toward  making  them  popular  for  medical  and  histological  work. 
His  Student  Stand  No.  2,  represented  by  Plate  VII,  is  a  very 


20  THE  MICROSCOPE   IN   BOTANY. 

efficient  instrument  of  this  class,  -a  solid  little  stand,  with  short 
body  and  limb,  a  draw-tube,  a  low  square  stage  with  included 
rotating  diaphragm,  and  a  heavy  horse-shoe  base.] 

[G.    THE  ILLUSTRATOR'S  STAND.] 

[Of  somewhat  erratic  model  is  the  microscope  called  the  Illus- 
trator's. It  is  made  by  T.  H.  McAllister  of  New  York,  and 
shown  in  Plate  VIII  B.  It  is  one  of  the  simplest  and  most  prac- 
tical of  those  designed  to  hold  several  objects  at  once.  It 
consists  of  a  broad  circular  base  from  the  center  of  which  rises 
a  pillar  that  carries  a  mirror,  a  circular  stage  25  cm.  in  diame- 
ter rotating  concentrically  about  the  pillar,  and  at  the  top  a 
horizontal  transverse  bar  with  its  vertical  compound  body.  The 
body  is  focussed  by  sliding  through  a  tube  in  the  transverse 
bar,  the  motion  being  controlled  by  a  pin  working  in  a  spiral 
slot.  The  stage  is  capable  of  carrying  twelve  slides,  radially, 
at  an  equal  distance  from  its  center,  which  can  be  successively 
brought  under  the  lenses  by  rotating  the  stage.  The  microscope 
is  best  adapted  to  large  objects  under  low  powers.  It  is  adapted 
to  certain  uses  in  teaching,  where  a  number  of  forms  are  to  be 
shown  in  comparison  with  each  other  to  a  class.  In  research, 
its  use  is  mainly  limited  to  the  rapid  comparison  of  objects,  as 
in  the  classification  of  unfamiliar  objects,  the  study  of  adulter- 
ations, or  the  comparison  of  samples  of  merchandise.] 

[H.    THE  HISTOLOQICAL  STAND.] 

[This  stand,  made  by  Mr.  Zentmayer,  and  represented,  with 
the  addition  of  the  Wenham  Binocular  arrangement,  Fig.  16,  in 
Plate  IX,  is  of  the  same  size  as  his  Student  Stand,  most  of  the 
castings  being  identical,  but  is  a  far  more  efficient  instrument. 
This  superiority  is  due  mainly  to  the  possession  of  asubstage,  a 
horizontal  ring  or  short  tube,  designed  to  support  the  diaphragm 
or  other  apparatus  that  may  be  required  between  the  stage  and 
the  mirror.  This  substage  is  carefully  centered  around  the  axis 
of  illumination  between  the  mirror,  in  whatever  position  it  may 
be  placed,  and  the  object  on  the  stage ;  and  it  has  a  smoothly 
sliding  vertical  movement  by  which  it  may  be  readily  located  at 


PL.  VII. 


Grunow's   (Continental)   Student  Stand. 


00 


co 


PL.   IX. 


Zentmaysr's  Histological  Stand. 


PL.  X. 


Bul loch's  Biological  Stand. 


PL.  XI. 


The   Bausch  and   Lomb  Universal  Stand. 


THE  UNIVERSAL   STAND.  21 

any  point  of  that  axis.  This  stand  is  made  with  a  glass  sliding 
st;ige,  or  with  a  round  rotating  stage,  if  desired.  In  the  monoc- 
ular form,  the  cost  may  be  reduced  by  substituting  for  the  rack 
and  pinion  coarse  adjustment  a  sliding  tube  like  that  in  Plate 
III.  It  is  most  commonly  made  monocular,  as  in  Plate  III,  but 
it  can  be  made  binocular  as  figured,  at  an  extra  cost;  as  can 
also  the  Acme  No.  3,  and  the  Biological  and  Universal.  It 
is  one  of  the  earliest  instruments  to  which  were  applied  several 
of  the  expedients  just  now  termed  the  modern  improvements 
of  the  microscope ;  and  it  presents,  in  combination  with  them, 
the  low  square  stage,  and  the  small  body,  of  the  continental 
style.] 

[I.     THE  BIOLOGICAL  STAND.] 

[Of  larger  instruments,  capable  of  utilizing  all  necessary 
accessories,  and  believed  by  the  writer  to  be  large  enough  for 
any  histological  work,  Mr.  Bulloch's  Biological  Stand,  repre- 
sented in  Plate  X,  was  one  of  the  first  to  assume  substantially 
its  present  form.  In  this  stand  the  tail-piece  is  made  double, 
one  portion  carrying  the  substage  and  the  other  the  mirror ;  an 
arrangement  which  is  essential  to  the  efficiency  of  this  modern 
device,  since  the  substnge  frequently  requires  to  be  in  a  position 
axial  to  the  compound  body,  for  the  purpose  of  holding  illumi- 
nating lenses  or  prisms,  for  instance,  at  the  same  time  that  the 
mirror  is  being  used  in  an  oblique  position.  The  usefulness  of 
the  whole  arrangement  is  impaired  in  such  cases  unless  the 
different  parts  can  be  moved  independently  of  each  other.] 

[J.     THE  UNIVERSAL  STAND.] 

[This  stand,  made  by  the  Bausch  and  Lomb  Co.,  is  repre- 
sented £  natural  size  in  Plate  XI.  It  comprises  the  same 
general  features  as  the  one  last  named,  but  by  a  slight  increase 
of  distance  between  the  stjige  and  the  table  sufficient  space  is 
secured  to  admit  the  use  of  the  largest  illuminating  or  polarizing 
apparatus,  etc.,  that  is  usually  employed  on  the  largest  stands. 
In  fact  there  is  scarcely  any  of  the  accessory  apparatus  of  the 
highest-priced  microscopes  that  cannot,  with  a  few  slight  modi- 


22  THE   MICROSCOPE   IN  BOTANY. 

fications  ill  non-essential  particulars,  be  easily  and  efficiently 
combined  with  this.  This  stand  can  be  obtained  as  shown  in 
the  cut,  in  a  very  simple  and  inexpensive  style ;  but  it  is  capa- 
ble of  a  much  higher  development.  It  has  been  constructed, 
for  the  use  of  the  writer,  with  the  addition  of  lengthening 
mirror  bar,  graduated  draw-tube  for  use  in  micrometry  and  in 
drawing  to  scale  at  any  desired  amplification,  centering  adjust- 
ment to  stage,  and  graduated  rotation  of  the  same,  centering 
substage  moved  vertically  with  rack  and  pinion,  and  graduated 
fine  adjustment  screw  with  index  point,  for  use  in  measuring 
.approximately  the  thickness  of  objects  or  cover-glasses.  It  is 
named  by  the  makers  the  "  Universal,"  from  the  belief  that  it  is 
possessed  of  the  working  capacity  of  the  most  elaborate  stands. 
The  stage  is  well  adapted  to  the  use  of  a  glass  sliding  stage  ; 
and  a  mechanical  stage  moved  in  all  directions  by  special  mech- 
anism can  be  added  if  desired.  R.  H.  W.] 

We  proceed  now  to  the  more  particular  consideration  of  the 
separate  parts  of  the  microscope  and  begin  with  the  most  im- 
portant part  of  the  optical  apparatus,  viz.,  the  objective. 


II.     THE  OBJECTIVE. 

The  objective  consists,  as  we  have  seen  on  p.  15,  of  several 
achromatic  double  lenses  joined  together  in  a  system.  The  ob- 
jective lenses  as  a  rule  are  plano-convex  and  are  formed  of  a. 
biconvex  converging  lens  of  crown  glass,  Fig.  1,  I  a,  and  a 
plano-concave  dispersing  lens  of  flint-glass,  Fig.  1,16.  The 

under,  convex  side  of  the 
former  corresponds  exactly  to 
the  upper,  concave  side  of  the 
latter.  The  two  are  cemented 
FIG.  i.i,  ir.  together  to  form  a  whole  by 

means  of  perfectly  transparent,  colorless  Canada  balsam.  Very 
rarely  and  only  in  glasses  made  for  the  greatest  magnification, 
the  underside  of  the  flint  glass  lens,  is  given  a  very  slight  con- 
cavity, Fig.  1,  II,  6,  so  that  these  in  the  same  manner  as  the 
others  become  an  achromatic,  concavo-convex  lens.  Formerly 


THE  ACHROMATIC  SET. 


23 


each  achromatic  lens  was  mounted  by  itself,  and  before  being 
used  several  were  combined  according  to  need.  This  was 
known  as  a  "set"  of  achromatic  lenses.  Now,  at  least  for 
the  larger  instruments,  these  achromatic  lenses  are  permanently 
combined  in  the  optical  manufactory  and  constitute  the  "ob- 
jective system." 

A.     THE  SET  OP  ACHROMATIC  LENSES. 

Each  achromatic  double  lens  is  mounted  in  a  short  brass  tube, 
as  is  shown  in  Fig.  2,  I,  II.  In  II,  d,  is  the  achromatic  lens 
with  the  plane  side  of  the  flint-glass  lens 
turned  downward.  It  is  placed  in  the  middle 
of  the  short  tube  in  the  upper  part  of  which 
is  cut  the  matrix  b,  into  which  the  whole  of 
the  patrix  of  a  similar  short  tube,  I  c,  may 
be  screwed.  Each  tube  is  designated  by  a 
successive  number,  and  the  lens  of  least  power 
bears  the  lowest  figure  and  that  of  the  greatest 
the  highest  figure.  In  III  is  represented,  for 
example,  a  set  of  lenses  belonging  to  an  old 
instrument  of  Schieck.  [Such  lenses  may  be 
used  singly  or  combined  into  sets  of  two  or 
three,  the  smallest  lenses,  when  the  size  varies, 
having  the  highest  power  and  being  placed  at 
the  bottom  of  the  combination.  R.  H.  TV.] 

These  sets  of  lenses  were  given  up  a  long 
time  ago,  and  are  applied  now  only  to  the  very  cheapest  instru- 
ments. In  scientific  botanical  investigations  we  shall  very 
seldom  be  in  a  situation  where  we  must  use  them.  T\  e 
consider  now  : 

B.     THE  OBJECTIVE-SYSTEM. 

Aside  from  the  advantages  already  mentioned  which  follow 
the  permanent  combination  of  several  double  lenses  into  a  sys- 
tem, certain  essential  improvements  in  respect  to  both  aberra- 
tions (see  pp.  5-6)  may  be  aimed  at  in  the  proper  arrangement 
of  the  lenses;  for,  as  Lister^  first  pointed  out,  the  special 

19  Lister  in  Philosophical  Transactions,  1S30,  p.  19S.  ff. 


24  THE  "MICROSCOPE   IN   BOTANY. 

aberration  of  double  lenses  may  be  made  to  compensate  each 
other  by  placing  them  at  proper  distances  apart.  It  is  not 
possible  to  correct  both  aberrations  perfectly,  for  the  difficulties 
attending  the  construction  of  very  small  achromatic  lenses  with 
short  foci  is  commonly  very  great,  the  grinding  of  the  most 
powerful  lenses  being  done  with  the  help  of  the  microscope  itself. 
Thus  the  best  systems  of  lenses  are  not  altogether  free  from 
faults,  but  these  faults  are  reduced  to  the  lowest  possible  limits. 
We  shall  just  here  briefly  mention  some  general  qualities  upon 
which  the  value  of  microscopical  glasses  depends  and  which  wTe 
already  in  the  foregoing  have  many  times  designated  by  name. 
There  are  two  principal  requirements  of  a  good  objective- 
system.  It  must  give,  first,  a  field  of  view  of  the  greatest  possible 
size  and  brightness,  and  second,  an  image  of  the  greatest  possi- 
ble distinctness. 

The  quantity  of  light  which  may  pass  through  a  lens  depends 
upon  its  superficial  area,  and  the  amount  of  light  which  may 
pass  through  two  lenses  of  different  sizes  is  proportioned  to  the 
square  of  their  diameters.  That  is,  a  lens  of  n  mm.  in  diameter 
will  afford  a  field  of  view  four  times  as  bright  as  one  |  mm.  in 
diameter.  The  degree  of  brightness  of  the  lens  is  measured  < 
by  its  "angle  of  aperture."  What  this  is  we  understand  by 
drawing  as  in  Fig.  3,  I,  straight  lines  from  two  diametrically 

opposite  points  on  the  edge 

.,          p        n       W      of  the  lens  g  h  to  the  focus 

B.      The  angle    EBF  in 
this  case  is  the  "  angle  of 
aperture."   It  is  divided  in 
halves  by  the  principal  axis 
of  the   lens   AB.™     In  a 
combination  of  lenses  the 
angle  of  aperture  will  not 
be   determined  by  the  ex- 
treme peripheral  rays  which  will  enter  the  front  lens  L  from  a 
luminous  point,  lying  in  its  focus  B,  II,  but  by  those  which  will 
pass  through  the  whole  combination  L  and  L'.      The  angle  of 

20  The  principal  axis  of  a  lens  is  that  straight  line  which  joins  the  middle  point  of  the 
spherical  surfaces  of  the  lens.  It  also  passes  exactly  through  the  middle  point  (o)  of  the  lens. 


THE   OBJECTIVE-SYSTEM.  25 

aperture  in  the  combination  illustrated  by  Fig.  3,  II,  is  not 
eBf  but  EBF.-1  From  this  it  follows  that  an  objective- 
system  which  shall  give  a  large  and  bright  field  of  vision22 
must  be  so  constructed  as  to  have  the  widest  possible  angle 
of  aperture.  But  it  has  been  found  in  practice,  that  there  is  a 
certain  maximum,  which  must  not  be  overstepped,  or,  in  the 
highest  magnifications,  the  image  will  be  materially  damaged  in 
other  respects. 

The  angle  of  aperture,  as  appears  in  the  foregoing,  is  great- 
est in  such  lenses  as  have  the  shortest  focus,  and  the  most  highly 
curved  surfaces.  But  here  again  conies  in  the  defect,  at  least 
if  it  have  a  spherical  surface,  which  we  have  several  times  des- 
ignated as  spherical  aberration.  Let  us  suppose  that  in  front 
of  a  lens,  Fig.  4,  in  its  principal  axis  AB  is  found  a  luminous 


FIG.  4. 

point  A.  This  emits  rays  Aa  Ab  Ac  Ad  on  the  upper  half, 
and  Ab'  Ac?  Ad'  on  the  lower  half  of  the  lens.  The  ray  Aa  falls 
perpendicularly  upon  the  surface  of  the  lens  in  the  direction  of  its 
principal  axis,  and  will  pass  through  unbent,  while  Ab  and  Ab1 
will  be  bent  in  a  definite  angle,  and  will  have  their  meeting  point 
behind  the  lens  in  B.  Likewise  Ac  and  Ac'  after  being  bent  will 
be  united  again  in  (7,  and  Ad  and  Ad'  in  D.  That  is  to  say,  the 
farther  removed  the  point  is  from  the  center  of  the  lens  through 
which  a  ray  passes,  the  more  it  will  be  bent,  and  the  shorter 

21  A  very  simple  contrivance  for  measuring  the  angle  of  aperture  of  a  microscopical 
objective-system  is  given  by  Dippel.  (Das  Mikroskop.  Braunschweig  1872,  Bd  I,  p.  86  f.). 

—  According  to  Dippel  (L  c.  p.  S3  ff.)  the  resolving  power  of  an  objective-system  depends 
upon  the  size  of  the  angle  of  aperture.  While  Hurting  (I.  c.  p.  219  ff.)  traces  this  back  to 
other  causes. 


26  THE  MICROSCOPE  IN  BOTANY. 

the  distance  behind  the  lens  where  they  will  be  reunited. 
We  get  three  points  behind  the  lens  where  the  rays  which  have 
been  emitted  from  A  come  together  again ;  viz.,  B,  C,  and  D. 
Nay,  many  more,  since  A  sends  out  not  only  the  rays  we  have 
supposed  but  an  infinite  number  of  others,  so  we  shall  have  an 
infinite  number  of  focal  points  all  lying  between  B  and  D.  We 
have  assumed  that  the  rays  Ab  and  AU  are  the  nearest  possi- 
ble to  the  principal  axis,  and  Ad  and  Ad1  the  nearest  possible 
to  the  edge  of  the  lens.  This  variation  in  the  focal  points  we 
call  the  spherical  aberration,  and  the  distance  B  to  Z),  the  length 
of  it.  If  now  we  hold  a  translucent  screen  or  a  piece  of  ground 
glass  at  the  point  B,  we  shall  see  thereon  a  picture  of  A,  but  it 
will  be  made  more  or  less  indistinct  by  the  rays  which  come 
from  the  outer  portions  of  the  lens,  and  have  crossed  and  been 
dispersed  at  C  D,  etc.  So  we  must  get  rid  of  these  so-called 
"marginal  rays"  Ac  Acf  Ad  Ad',  etc.,  by  shutting  them  off. 
This  can  very  easily  be  done  by  means  of  a  diaphragm.  To 
accomplish  this,  all  that  is  necessary  is  to  place  an  opaque  disk 
with  a  circular  hole  in  the  middle  of  it  between  the  lens  and 

Q 

A,  or  the  lens  and  B,  so  as  to  permit  only  the  middle  rays  Aa 
Ab  Ab',  etc.,  to  pass  through.  This  is  indeed  a  very  simple 
method  of  overcoming  the  spherical  aberration,  but  it  is  only  to 
a  certain  extent  satisfactory,  since  naturally  what  such  a  dia- 
phragm shuts  off,  reduces  the  angle  of  aperture  by  so  much,  and 
in  consequence  thereof,  the  field  of  vision  will  be  made  smaller 
and  its  brightness  much  diminished.  It  is  on  this  account,  of 
the  greatest  importance,  that  the  spherical  aberration  should  be 
reduced  in  some  other  way,  viz. ;  by  the  peculiar  construction  of 
the  lens  itself. 

If  a  lens  be  not  alike  convex  on  both  sides,  but  if  one  side 
have  less  curvature  than  the  other,  or  the  lens  be  plano-convex 
or  plano-concave,  its  spherical  aberration  will  be  diminished,  as 
experience  teaches,  if  the  side  of  least  curvature,  the  plane  or 
concave  side  of  the  lens  be  turned  towards  the  object  (at  least 
with  microscopical  objectives).23  It  has  been  shown  that  those 
biconvex  lenses,  which  have  surfaces  of  unequal  curvature  and 

23  With  telescopic  lenses,  whose  object  is  very  far  removed,  we  must  reverse  this  rule 
and  present  that  side  of  the  lens  which  has  the  greatest  curvature  to  the  object. 


THE   OBJECTIVE-SYSTEM.  27 

the  least  spherical  aberration  are  those  whose  curvature  on  one 
side  has  a  radius  six  times  as  long  as  that  of  the  other.24 

The  spherical  aberration  is  diminished  also,  by  combining  a  bi- 
convex lens  of  crown  glass,  and  a  plano-concave  lens  of  flint  glass. 
In  a  biconvex  converging  lens  the  spherical  aberration  is  of  that 
nature  that  the  marginal  rays  come  to  a  focus  in  front  of  the 
true  focus  of  the  lens.  A  diverging  plano-concave*  lens  has  an 
aberration  of  like  nature  only  with  this  important  difference,  that 
the  marginal  rays  come  to  a  focus  behind  the  focus  of  the  central 
rays,  or  the  true  focus  of  the  lens.  If  now  the  two  lenses  be 
made  of  glass,  of  different  refractive  powers,  and  then  combined 
to  make  a  whole,  and  rays  of  light  be  passed  through  them,  the 
relative  direction  of  the  central  and  marginal  rays  will  be  reversed 
by  the  action  of  the  diverging  lens.  It  comes  about,  thence, 
that  by  the  united  effect  of  the  two  lenses  the  focal  point  of  all 
the  rays  will  be  brought  very  near  together ;  only  it  must  be 
understood  that  the  lenses  should  be  made  with  a  definite  curva- 
ture on  their  contiguous  sides,  which  indeed  we  need  not  here 
more  particularly  specify. 

Finally,  there  is  another  very  effective  means  for  materially 
lessening  the  spherical  aberration,  which  consists  in  uniting  three 
plano-convex  doublets  in  one  system.  In  this  we  follow  Lister,25 
who  discovered  the  one  peculiar  characteristic  of  the  achromatic 
doublets,  and  gave  formulas  for  their  combination  based  on  this 
discovery.  The  distance  apart  of  the  three  lensete  must  be  a 
very  definite  one.  It  is  the  art  of  the  optician  by  exact  and 
most  careful  experimentation  to  give  the  lenses  their  right  posi- 
tion in  his  objective-system.  The  smaller  the  achromatics,  the 
shorter  their  focal  distance  and  the  more  difficult  is  it  to  accom- 
plish this  combination.  It  is  for  this  reason  that  good  object- 
ive-systems are  so  expensive.  The  great  advantage  which  is 
attained  by  this  combination  of  three  lenses  is  that,  notwithstand- 
ing the  great  magnifying  power  of  the  lenses  singly,  and  in  spite 
of  correcting  their  spherical  aberration,  the  angle  of  aperture 
is  still  large  and  the  field  of  vision  still  shows  great  brightness. 

24  This  proportion  answers,  however,  only  for  those  kinds  of  glass  which  have  an  index 
of  refraction  of  1.5. 

25  See  more  particularly  Lister,  at  the  place  before  mentioned  and  in  llarting,  /.  c.,  p. 
46  f,  and  138  ff. 


28  THE  MICROSCOPE  IN  BOTANY. 

It  is  a  generally  known  fact  that  by  uniting  a  biconvex  crown 
glass  lens  with  a  plano-concave  flint  glass  lens  the  chromatic 
aberration  of  the  rays  is  almost  entirely  overcome.  We  have 
already  stated  the  reasons  for  this  on  page  5.  We  believe  that 
in  this  cursory  review  of  the  physics  of  the  matter  we  shall  not 
be  expected  to  go  into  it  more  particularly.26 

We  will  therefore  only  add  in  brief,  that  in  refraction  through 
a  single  lens,  the  violet  rays  have  their  focus  nearest  the  lens, 
are  the  most  converged,  while  the  focus  of  the  red  rays  is  farthest 
removed  from  it.  The  focal  points  of  all  the  other  colored  rays 
lie  between,  naturally,  in  the  order  in  which  they  appear  in  the 
spectrum.  An  achromatic  lens-combination  is  now  so  corrected 
that  with  it  the  focal  distance  for  red  and  violet  light  is  the 
same.  In  this  way  these  two  colors  of  the  spectrum  fall  upon 
the  same  point,  but  not  so  with  those  which  lie  between.  These, 
as  is  easily  seen,  give  secondary  dispersion  images  which  appear 
as  colored  borders  of  different  tints,  commonly  yellow  or  green. 
To  remove  these  it  is  only  necessary  to  make  a  combination  of 
several  doublets,  and  in  fact  this  requirement  is  met  in  a  combi- 
nation such  as  is  the  present  objective- system.  As  in  these 
systems  the  spherical  aberration  is  reduced  to  its  lowest  terms, 
so  also  the  chromatic  aberration  is,  for  the  most  part,  overcome. 
At  the  present  time,  if  the  flint-glass  lens  is  given  a  very 
slight  preponderance  the  result  is  that  the  system  shows  a  very 
soft,  and  to  the  eye  a  very  pleasant  blue  tinge,  in  the  micros- 
copic image.  We  call  such  a  lens  as  that  over-corrected.  If 
the  lens  on  the  other  hand  shows  a  red  border  (by  reason  of  a 
preponderance  of  crown  glass)  we  say  it  is  under-corrected. 

Since  the  objective-system  is  capable  of  all  the  wished-for 
corrections,  of  which  we  have  spoken,  we  have  in  it  what  we 
call  an  "aplanatic,"  that  is,  a  lens  with  the  least  possible  error, 
the  smallest  amount  of  both  spherical  and  chromatic  aberration. 


The  Systems  in  Practice.     Now  that  we  have   become  ac- 
quainted with  the  optical  principles  of  the  objective-systems, 

26  See  thereon  for  example  Wullner's  Lelirbuch  der  Experimentalpliysik,  Leipzig,  1871, 

Bd.  II,  p.  21(5-2-20 v.  Quiiitus-Icilitis.     Experimentalphysik,  Hanover,  1SUG,  p.  250ff. 

Hurting.  I,  c.,  p.  37-40. 


THE  OBJECTIVE-SYSTEM.  29 

we    shall  proceed  to    consider  some  of  the  different  sorts   of 
objectives. 

The  common  system  consists  of  three  achromatic  doublets  of 
the  form  shown  in  Fig.  1,  I.  They  are  arranged  as  is  shown  in 
Fig.  5,  a,  £>,  c.  The  smallest,  which  is  also  the 
strongest  magnifier,  is  nearest  the  object ;  the 
largest  and  weakest  is  removed  farthest  from  it. 
By  this  combination  there  is  attained  on  the  one 
side  a  greater  focal  distance  of  the  object,  and 
on  the  other  an  objective  so  constructed  gives  a 
wide  angle  of  aperture  and  a  very  bright  image. 
Differing  from  this,  a  very  perfect  system  is 
sometimes  made  by  combining  an  under  lens  of  three  parts,  a 
plano-concave  of  flint  glass,  and  two  plano-convex  lenses  of 
crown  glass,  with  a  middle  lens  of  the  form  of  Fig.  1,  I,  and 
an  upper  lens  constructed  of  a  middle  bi-concave  of  flint  glass 
and  two  bi-convex  lenses  of  crown  glass. 

The  mounting  of  an  objective-system  is  clearly  represented 
in  Fig.  6,  which  is  a  medium  system  of  natural  size  from  the 
manufactory  of  Seibert.  The  three  plano-convex  doublets  are 
contained  in  the  lower  half  cZa,  which  part  is  screwed  into  the 
upper  db,  the  latter  having  no  lenses.  Both 
parts  are  made  permanently  fast  to  each  other. 
At  b  the  system  is  provided  with  a  screw  thread 
by  which  it  is  made  fast  to  the  microscope.  In 
this  manipulation  one  takes  it  with  the  thumb, 
index  and  middle  fingers  of  the  right  hand,  on 
the  two  edges  cc,  their  milling  enabling  one  to 
hold  it  fast.  On  the  smooth  surface  e  between 
the  two  edges  the  number  [and  maker]  of  the 
system  is  engraved.  [Somewhat  similar  ob- 
jective-svstems  are  shown  in  situ  at  the  bottom  of  the  mi- 
croscope tube,  or  compound  body,  in  Plates  IV  and  VI  to  XI. 
R,  H.  AV.] 

The  use  of  such  a  system  (the  so-called  dry  lens)  is  very 
easily  understood.  But  the  use  of  two  other  systems  of  con- 
struction, mainly  applied  to  the  production  of  much  higher 
magnifications  ;  viz.,  the  "immersion-system"  and  the  "correction- 
system,"  is  very  much  more  elaborate. 


30  THE   MICROSCOPE  IN  BOTANY. 

The  Immersion- System.  In  order  to  understand  this  system, 
it  is  necessary  to  assume  that  the  botanical  object  to  be  viewed 
by  the  microscope  lies  under  a  thin  glass  plate  —  the  cover- 
glass —  and  is  surrounded  by  a  layer  of  water  or  other  fluid, 
glycerine,  essential  oils,  etc.  The  light,  which  enters  the  optical 
apparatus  of  the  microscope  from  the  object  has  to  pass,  on  its 
way,  through  one  after  another  the  several  media,  water,  glass, 
air.  Since  the  glass  and  the  air  are  very  different  in  their  re- 
fractive power,  and  the  light  enters  from  a  thicker  medium,  glass, 
into  a  thinner,  air,  a  number  of  the  rays  will  be  so  far  dispersed 
that  they  will  not  be  able  to'  enter  the  objective-system,  and 
the  consequence  will  be  that  the  microscopic  image  will  be 
correspondingly  darker  ;  and  further,  a  considerable  reflection  of 
the  rays  of  light  will  take  place  from  the  under  plane  surface  of 
the  lens. 

In  order  to  remove  this  defect  we  have  for  a  long  time  adopted 
this  contrivance,  with  high  magnifying  powers,  of  substituting 
a  thin  film  -  of  water  for  the  layer  of  air  which  intervenes 
between  the  cover-glass  and  the  front  lens  of  the  objective. 
We  owe  this  especially  to  Hartnack.  Since  the  refractive 
power  of  the  water  is  much  nearer  that  of  glass  than  is  that  of 
air,  it  is  obvious  that  the  interposition  of  the  film  of  water  will 
very  considerably  diminish  the  before-mentioned  dispersion  of 
the  rays  of  light,  and  will  cause  therefore,  many  more  to  enter 
the  objective  and  will  give  to  the  microscopic  image  a  consider- 
ably greater  brilliancy.  The  result  of  this  arrangement  is 
essentially  the  same  as  if  the  angle  of  aperture  of  the  objective 
were  considerably  increased.  By  this  means  the  reflection  of 
the  rays  from  the  under  surface  of  the  lens  and  also  from  the 
upper  surface  of  the  cover-glass  is  altogether  obviated.  Such 
a  system,  whose  lowest  lens  is  immersed  in  water  is  called  a 
"water-system"  or  "immersion-system."  This  objective  is 
prepared  for  use  in  the  following  way.  The  objective  is  turned 
bottom  side  up,  and  a  drop  of  distilled  water  from  a  flask,  by 
means  of  a  glass  rod  or  hair  pencil,  is  placed  upon  the  lens. 
Here  it  will  round  itself  up  into  a  little  hemisphere.  Now 
reverse  the  objective  and  the  drop  will  remain  in  place  by  the 
power  of  adhesion.  The  objective  should  now  be  screwed  into 


THE  IMMERSION-SYSTEM.  31 

the  microscope  tube,  and  the  tube  pushed  down  by  hand  or  by 
the  rack  and  pinion  till  it  comes  near  the  cover-glass  upon  the 
object  which  lies  upon  the  stage.  Now  if  one  breathes  a  little 
upon  the  cover-glass  and  then  carefully  brings  the  drop  down 
till  it  touches  it,  it  will  easily  unite  with  the  surface  of  the 
glass  and  form  the  desired  film  of  water  between  the  glass  and 
lens.  Then,  by  means  of  the  fine  adjustment  screw,  the  object 
can  be  exactly  focussed.  Particular  care  should  be  taken  that 
the  drop  of  water  used  does  not  contain  even  the  smallest  bubble 
of  air,  else  the  microscopic  image  will  be  ruined.  After  use,  the 
drop  of  water  which  adheres  to  the  objective  should  be  carefully 
wiped  off  with  a  piece  of  soft  old  b'nen  cloth  which  has  been 
washed  in  distilled  water. 

[It  is  sometimes,  moreover,  convenient  to  plunge  the  objec- 
tive directly  into  the  medium  in  which  objects  are  situated,  for 
the  purpose  of  examining  them  without  preparation  or  selection, 
and  under  strictly  natural  conditions.  When  the  medium  is 
not  corrosive,  ordinary  "immersion"  objectives  may  be  used 
in  this  manner,  provided  they  have  sufficient  screw-collar 
movement  to  make  the  necessary  corrections ;  and  objectives 
specially  corrected  for  this  use  have  been  constructed  by  Tolles 
and  others.  Such  lenses  thus  used,  though  not  inapplicable  to 
certain  botanical  researches,  have  been  heretofore  mostly  em- 
ployed in  zoology  and  pathology.  By  a  modification  of  this 
plan,  however,  dry  lenses  of  lower  powers  may  acquire  a  new 
value  to  the  botanist.  By  surrounding  the  objective  with 
a  brass  cylinder  open  above  and  closed  tightly  with  a  thin 
cover-glass  below,  it  may  be  plunged  into  water  or  various 
solutions,  with  impunity,  and  a  clear  and  satisfactory  view  may  be 
obtained  of  objects  in  the  fluid,  or  lying  at  its  bottom,  in 
saucers,  dissecting  troughs,  or  other  suitable  vessels.  Of  course 
the  vessels,  or  their  bottoms,  must  be  of  glass  if  transmitted  light 
be  required.  By  this  method,  not  only  the  unavoidable  tremor 
of  the  upper  surface  of  the  liquid,  which  renders  study  of  objects 
below  by  usual  methods  difficult  and  quite  unsatisfactory,  is  ren- 
dered harmless ;  but  the  object  may  be  freely  manipulated  with 
needles,  scissors,  or  dipping  tubes,  under  objectives  of  from  two 
inches  (51  mm.)  to  a  low-angled  J  or  £,  without  interrupting  the 


32 


THE   MICROSCOPE   IN   BOTANY. 


FIG.  7. 


view.  Small  objects,  in  small  quantities,  may  be  thus  examined 
under  the  higher  powers  in  watch-glasses.  For  larger  quanti- 
ties and  lower  powers,  nothing  is  more  convenient  than  the  little 
glass  dishes  occasionally  sold  as  individual  butter  plates,  or  the 
glass  jars  sold  as  seed  and  drink  cups  for  bird 
cages.  WThen  the  quantity  of  material  is 
unlimited,  and  especially  when  manipulation 
is  required,  nothing  is  more  convenient  than 
plain  glass  preserve  dishes  one  inch  (25  mm.) 
deep,  and  four  inches  (10  cm.)  wide.  For 
dissecting  purposes  with  reflected  light  ex- 
clusively, china  dishes,  or  the  hard  rubber  dissecting  troughs 
sold  by  microscope  dealers,  may  be  used ;  the  bottoms  being 
lined  with  thin  sheets  of  cork  if  it  be  desired 
to  fasten  down  the  objects  with  pins.  A  most  ^_J  \_ 

convenient  apparatus  for  this  quasi-immersion 
use  of  dry  lenses  is  the  "  objective-protector," 
Fig.  7  ;  shown  in  section  and  in  situ  upon  the 
objective  in  Fig.  8.  It  was  proposed  by  Mr. 
R.  E.  Dudgeon  of  London,  and  is  made  by  J. 
H.  McAllister  of  New  York.  R.  H.  W.] 

The      Correction- System.       We    will    now  FlG- 8- 

suppose  that  an  object  Fig.  9,  p,  lies  on  the  microscope,  under 
the  cover-glass  DD  (a  highly  magnified  section  in  the  illus- 
tration) through  which  we 
send  rays  of  light  pv,  pa, 
p(',pe,  .  .  .  pa',  pcr,  pe', 
from  the  illuminating  mirror, 
to  the  objective.  The  rays 
impinge  upon  the  under  sur- 
face of  the  cover-glass,  and 
those  which  fall  upon  it  ob- 
liquely do  not  proceed  in 
the  same  direction  as  here- 
tofore, but,  at  the  surface  between  the  water  and  the  glass, 
are  refracted  and  within  the  glass  take  the  direction  a  A,  cC,  eE, 
.  .  .  .a'A^c'C^e'E'.  Further,  in  passing  through  the  upper  surface 
of  the  cover-glass  into  the  air  or  into  the  water,  as  the  case  may 


THE  CORRECTION-SYSTEM.  33 

be,  in  dry  or  immersion  objectives,  they  are  again  refracted  in 
the  direction  AF,  CG,  EH,  .  .  .  A'F',  C'G',  EH.  The 
rays  of  light  are  more  widely  dispersed  on  emerging  in  propor- 
tion to  the  acuteness  of  the  angle  at  which  they  enter  the  cover- 
glass  from  p.  If  we  now  construct  the  uniting  points  of  the  twice 
refracted  rays  we  shall  have  a  series  « — s  along  the  line^v,  one 
point  above  another,  each  of  which  represents  the  image  of  the 
preparation  p,  as  a  luminous  point.  The  distance  a  —  swill 
be  less  or  greater  in  proportion  to  the  thickness  of  the  cover- 
glass  DD.  There  results  from  this  exactly  the  same  phenome- 
non that  we  have  learned  to  know  as  spherical  aberration. 
The  use  of  the  cover-glass,  therefore,  will  cause  a  certain 
indistinctness  of  the  microscopic  image  similar  to  that  caused 
by  the  use  of  an  objective  which  has  not  been  corrected  for 
spherical  aberration.27 

We  have  alreddy  learned  that  the  defect  of  spherical  aberra- 
tion can  be  corrected  by-placing  the  three  objective' lenses  in  a 
certain  right  relative  position  to  each  other.  It  is  also  evident, 
that  the  optician  can  likewise  eliminate  the  damaging  influence 
of%  the  cover-glass,  if  the  same  thickness  of  cover-glass  were 
always  used,  and  he  had  taken  into  account  this  particular  thick- 
ness in  correcting  the  objective  for  spherical  aberration.  This 
is  now  almost  always  done  and  the  objectives  for  medium  mag- 
nifications are  commonly  so  corrected  that  they  give  the  clearest 
possible  image  with  the  use  of  a  cover-glass  .1  to  .2  mm.  thick. 

For  high  power  objectives,  chiefly  for  strong  immersion 
lenses,  with  which  the  influence  of  the  cover-glass  is  most  dam- 
aging to"  the  clearness  of  the  image,  we  have  followed  Ross28 in 
hitting  upon  a  contrivance  which  almost  entirely  eliminates  this 
bad  influence,  viz.,  a  device  for  changing  at  will  the  relative 
distance  of  the  lenses,  and  thus  obtain  an  objective  which  is 
aplanatic  for  every  thickness  of  cover-glass.  We  name  this 
the  "  adjustable  lens,"  the  "  correction  system,"  the  "  system  with 
correction  for  thickness  of  cover-glass." 

27  More  particularly  H.  v.  Mohl,  I.  c.',  p.  157/.— Harting,  I.  c.,  p.  146-149. 

28  Ross  discovered  this  influence  of  the  cover-glass  in  1837  and  sought  at  once  to  obviate 
it  by  the  screw  correction  in  the  system  (Harting,  1.  c.,  p.  747).    Already  before  this  (1829) 
Amici  had  made  the  same  discovery,  but  constructed  however,  no  correction,  system,  but 
added  to  his  microscopes  several  equivalent  systems  which  were  intended  for  different 
thicknesses  of  cover-glass  (Harting,  I.  c.,  pp.  148,  720). 


34 


THE  MICROSCOPE   IN  BOTANY. 


Ross  so  constructed  his  objectives  that  the  correction  was 
made,  by  changing  at  will,  by  means  of  a  screw  contrivance, 
the  distance  between  the  upper  lenses  which  Avere  fastened 
together  as  a  whole,  and  the  lower  lens.  Afterwards  Hartnack 
modified  this  so  that  the  two  lower  lenses  which  were  made  fast 
together  were  made  movable  towards  the  upper  which  was  fixed 
in  the  microscope  tube.  Finally,  several, 
especially  German  makers,  have  modified  the 
system  of  Hartnack  so  that  the  two  under 
lenses  are  made  fast  together  and  to  the  mi- 
croscope tube,  and  the  upper  lens  is  mounted  in 
an  inner  movable  sheath  so  as  to  be  adjusted  to 
the  other  two.  The  last  two  methods  of  con- 
struction do  not  ditler  at  all  in  regard  to  the 
results  produced,  and  the  difference  between 
Ross'  and  Hartnack's  system  of  correction  is  of  a  subordinate 
nature.  In  Fig.  10  is  represented  a  longitudinal  section  through 
a  Hartnack  objective  which  shows  the  characteristics  of  the  ad- 
justment. By  means  of  the  screw  thread  b  the  inner  cylinder  i 
which  bears  the  upper  lens  3  is  made  fast  to  the  microscope 
tube.  The  two  under  lenses  1  and  2  are  screwed  fast  to  the  outer 
cylinder  a  into  which  the  inner  shell  i  exactly  fits.  At  c  is  a 
ring  which  by  means  of  a  screw  thread  can  be  moved  up  and  down 
on  i.  This  ring  bears  in  an  inner  groove  v 
the  cylinder  a.  Now  if  the  ring  be  screwed 
up,  the  tube  a  with  the  lenses  1  and  2  will 
be  carried  up  and  not  at  the  same  time  turned 
around  the  axis  of  the  objective  with  the  ro- 
tary motion  of  the  ring. 

In  reference  to  manipulating  the  correction 
in  microscopical  work  the  following  may  be 
said.  Fig.  11  represents  the  immersion  No. 
VII  of  Seibert,  natural  size.  The  objective 
has  a  screw  collar  c  corresponding  to  c  in  Fig.  10,  which  on  its 
upper  smooth  part  is  garduated  into  ten  divisions  (1,  2,  3,  etc.) 
which  graduations  play  by  a  mark  at  e.  By  a  full  turn  of  c  the 
upper  lens  is  removed  from  or  brought  nearer  to  the  lower 
lenses,  as  the  case  may  be,  by  the  distance  of  a  screw  thread. 


FIG.  11. 


THE   OCULAR.  35 

The  interval  between  the  graduations  corresponds  to  .1  of  this 
change  of  distance.  Now  to  enable  one  to  control  this  move- 
ment of  the  upper  lens  up  and  down  from  a  certain  medium 
position  the  device  a  i  has  been  contrived.  A  small  slit  is  cut 
through  the  outer  cylinder  a  which  allows  the  inner  cylinder  i 
to  be  seen  through  it.  Both  a  and  i  are  provided  with  a  mark. 
If  they  stand  at  the  same  height  (as  they  do  not  in  the  illustra- 
tion) then  the  upper  lens  is  in  a  normal  position  to  the  under. 
If  the  middle  line  i  is  above  the  other  a  the  upper  lens  is 
moved  farther  away  from  the  lower,  and  the  reverse.  After 
the  lenses  have  been  put  in  their  normal  position  by  bringing  the 
0  of  the  ring  graduations  to  the  mark  e,  the  objective  should  be 
put  on  to  the  microscope,  focus  the  object  and  then  turn  the 
ring  experimentally  back  and  forth  till  the  image  becomes  very 
sharp  and  distinct.  When  this  is  done  a  note  should  be  made 
on  the  slide,  of  the  direction  and  distance  which  the  ring  has 
been  moved. 


HI.     THE   OCULAR. 

We  have  already  seen  on  p.  15  that  the  ocular  consists  of 
two  glasses,  the  upper  one  being  placed  near  the  eye  and  is  the 
essential  image-viewer,  and  in  the  narrow  sense  the  ocular-glass, 
while  the  under  one  called  a  "  collecting 

o 

lens,"  is  only  in  a  limited  sense  a  part 
of  the  ocular  and  might  with  much  greater 
propriety  be  considered  a  part  of  the  ob- 
jective. But  since  they  are  always  united 
to  make  this  part  of  the  microscope  it 
has  always  been  customary  to  designate 
the  two  glasses  together  as  the  ocular. 

Fig.  12,  which  represents  a  somewhat 
conventional    longitudinal    section    of  a 
Hartnack  ocular,  will  enable  us  to  under- 
stand the  arrangement  of  oculars.    In  a  FlG  1§ 
cyb'ndrical  brass  shell,  which  exactly  fits 

into  the  tube  of  the  microscope,  there  are  screwed  two  end 
pieces,  one  c  above,  and  the  other  d  below,  of  which  the  one 


36  THE  MICROSCOPE   IN   BOTANY. 

carries  the  ocular  lens  at  a  and  the  other  the  collecting  lens 
at  b.  Inside  of  e  at  about  equal  distance  from  a  and  b  is 
placed  the  diaphragm  /,  intended  to  cut  off  the  marginal  rays 
coming  through  6,  which  are  so  deleterious  to  the  beauty  of  the 
image.  Both  lenses  have  a  plano-convex  form,  and  the  convex 
side  of  both  is  downward.  This  arrangement  of  the  lenses  with 
their  curved  sides  down,  essentially  influences  the  size  of  the 
field  of  view  and  the  sharpness  of  the  image.  A  reversal  of 
them  would  materially  diminish  the  field  as  well  as  the  sharp- 
ness and  flatness  of  the  image. 

The  ocular  is  set  in  the  top  of  the  microscope-tube.  The  oc- 
ular lenses  should  be  exactly  centered  with  the  objective  lenses, 
that  is,  so  made  that  a  straight  line  drawn  through  the  middle 


FIG.  13.  FIG.  14. 

point  of  the  objective  lenses  shall  also,  on  being  prolonged,  pass 
through  the  middle  point  of  the  ocular  lenses.  This  presup- 
poses exact  workmanship.  Oculars  have  the  form  of  a  simple 
cylinder  Fig.  13,  or  in  later  times  that  represented  in  Fig.  14. 
The  first  is  found  in  the  microscopes  of  Hartnack,  Merz  and 
Nachet,  and  the  second  in  those  of  Gundlach  and  Seibert  [and 
?n  those  of  most  English  and  American  makers.  R.  H.  W.] 
That  part  of  the  ocular  which  slips  into  the  microscope  tube 
should  exactly  and  easily  fit  into  it.  In  putting  the  ocular  into 
the  microscope-tube  it  should  always  be  allowed  to  sink  down  to 
cc  Figs.  13,  14,  in  the  tube.  The  distance  of  the  ocular  from 


THE   OCULAR. 


37 


the  objective  is  a  definite  one  and  should  not  be  changed  at  will, 
save  in  those  cases  to  be  described  farther  on  in  treating 
w  microscope-tubes." 

For  the  ocular  is  placed  at  such  a  distance  from  the  objective 
that  the  image  produced  by  it  would  appear  above  the  collect- 
ing lens.  In  other  words  the  collecting  lens  is  below  the  point 
where  the  cone  of  refracted  rays  from  the  objective  would 
meet. 

If  we  suppose  a  cone  of  refracted  rays  of  a  magnified  object, 
from  the  objective,  to  be  represented  by  a  A,  bB,  cC,  dD,  eE, 
b'B\  cfC',  d'Z>',  4E1,  Fig.  15,  it  would  present  itself  to  us  in 


e  d  c  ba  b'c'd'e 

.    Flo.  1.3. 

the  extended  form  represented  by  EE1.  Now  we  shove  down 
beneath  this  image  the  collecting  or  "field  lens,"  LL,  corres- 
ponding to  b,  Fig.  12,  and  the  rays  which  fall  upon  it  at  fc,  r, 
fl,  Jf,  fo',  jp',  (T,  t't  will  be  refracted  inward  toward  the  axis  aA, 
and  take  the  direction  fcg,  dfc,  4g,  *tf,  fc'§',  *'$',  A'g7,  t'd7, 
the  diverging  bundle  of  rays  changed  to  a  converging,  and  the 
image  thus  modified  by  the  field  lens  will  fall  at  $  (&'.  The 
collecting  lens  has  really  diminished  the  image.  But  this  loss 
of  magnification  is  in  various  ways  an  advantage,  for  now  more 
of  the  image  can  be  seen  than  when  the  rays  are  diverging.  It 
is  also  evident  that  the  interposition  of  the  field  lens  increases 


38  THE   MICROSCOPE   IN  BOTANY. 

the  brightness  of  the  image  by  concentrating  the  given  number 
of  rays  upon  a  smaller  surface.  In  like  manner  the  distortion  of 
the  image  by  the  unequal  magnification  of  the  central  and  mar- 
ginal portion  is  obviated.  To  this  end  also  the  diaphragm 
mentioned  above  affords  no  small  help  by  cutting  off  most  of 
the  distorting  marginal  rays. 

The  effect  of  the  ocular  glass  a  Fig.  12,  is  that  of  a  simple 
magnifying  glass,  by  which  the  picture  at  $  $'  is  enlarged.  It 
is  not  necessary  to  mention  that  the  ocular  lens  should  be  so 
placed  that  the  image  $  $>'  will  be  exactly  in  its  focus,  or  that 
combining  the  ocular  and  field  glass  with  the  objective  will 
materially  assist  in  correcting  its  spherical  and  chromatic  aber- 
ration. They  can  be  adjusted  by  somewhat  over  correcting  the 
aberrations  of  the  objective  and  somewhat  under  correcting  those 
of  the  oculars.  The  oculars  of  most  microscopes  have  the 
arrangement  just  now  described.  It  is  called  the  Campanian 
or  Huygenian,  ocular  (negative  ocular).  This  is  at  the  present 
time  sometimes  so  altered  that  the  field  lens  is  made  of  a  con- 
cavo-convex flint-glass  and  a  biconvex  crown-glass  cemented 
together  with  Canada  balsam,  thus  making  a  biconvex  achro- 
matic doublet.  This  modification  of  the  Huygenian  ocular  was 
first  made  by  Kellner  and  was  called  the  orthoscopic  ocular. 
The  aplanatic  ocular  of  Plossl  is  much  the  same  thing,  made  of 
two  achromatic  plano-convex  lenses,  and  are  combined  for  the 
most  part  as  shown  in  Fig.  12. 

A  very  useful  variation  is  found  in  the  Kamsclen  or  positive 
ocular.  The  lenses  have  the  same  form  as  in  Fig.  12,  but  the 
field  lens  is  permanently  reversed,  turned  with  its  plane  side 
toward  the  objective,  while  its  distance  from  the  ocular  lens  is 
much  less  than  in  the  Huygenian  oculars.  For  common  obser- 
vations the  positive  ocular  is  seldom  used.  It  is  mainly  useful 
in  fine  microscopical  measurements,  but  is  not  absolutely  neces- 
sary even  for  these. 

[IV  THE  BINOCULAR  OCULAR.] 

[For  the  sake  of  the  advantages  of  stereoscopic  vision,  and 
of  the  comfort  secured  by  using  both  eyes  instead  of  one,  the 


THE  BINOCULAR  OCULAR.  39 

pencil  of  rays  above  the  objective,  or  above  an  erector  inserted 
within  the  draw-tube,  is  sometimes  divided  into  two  portions, 
one  of  which  is  transmitted  to  each  eye.  When  the  apparatus 
is  permanently  attached  to  a  modification  of  the  compound 
body,  it  is  termed  a  binocular  microscope,  and  when  mounted 
separately  and  capable  of  removal  like  simple  oculars,  it  is 
called  a  binocular  ocular,  or  eye-piece.  Among  those  who, 
when  the  stereoscope  was  an  interesting  novelty,  undertook  to 
apply  its  principles  to  the  microscope,  the  first  to  succeed  was 
Prof.  J.  L.  Riddell  of  Now  Orleans,  La.,  who  is  therefore 
justly  credited  with  the  invention  of  the  binocular  microscope. 
He  bisected  the  pencil  of  rays  above  the  objective  by  a  pair  of 
rectangular  prisms  which  turned  the  parted  halves  of  the  pencil, 
by  internal  reflection,  horizontally  across  each  other  to  a  dis- 
tance apart  equal  to  the  distance  from  each  other  of  the  pupils 
of  the  eyes,  at  which  points  they  were  again  reflected  directly 
upwards  by  a  second  pair  of  prisms  to  the  two  oculars.  Shortly 
afterwards,  Nachet  of  Paris  slightly  modified  aud  improved 
this  plan,  dividing  and  crossing  the  rays  by  internal  reflection 
from  the  opposite  sides  of  a  single  equilateral  triangular  prism, 
the  rays  passing  thence  to  a  pair  of  prisms  below  the  oculars  in 
a  direction  not  horizontal  but  inclined  upward.  Not  long  after- 
wards, the  late  R.  B.  Tolles  of  Boston,  still  further  improved 
this  apparatus  by  transferring  the  whole  system  of  prisms  to  a 
position  just  below  the  oculars  and  above  an  erector  attached 
to  the  lower  end  of  the  -apparatus,  the  whole  being  removable 
together,  and  leaving  the  tube  ready  for  the  reception  of  any 
simple  ocular.  Notwithstanding  its  excellent  workmanship, 
good  definition,  fine  stereoscopic  effect,  ease  of  removal,  and 
applicability  to  all  powers  however  high,  this  ocular  never  came 
into  use,  less  perhaps  on  account  of  its  considerable  cost,  than 
because  of  the  small,  high  power  oculars  and  parallel  tubes 
required  in  its  construction.  Oculars  of  high  power  are  tire- 
some to  the  eyes  at  best,  and  especially  when  the  light  has  also 
passed  through  the  numerous  refracting  and  reflecting  media 
composing  an  erector  and  set  of  reflecting  prisms ;  and  to  pre- 
serve the  parallelism  of  the  axis  of  the  eyes  as  required  for  dis- 
tant vision,  when  looking  intently  at  an  object  known  to  be 


40 


THE   MICROSCOPE   IN   BOTANY. 


near,  is  a  continual  strain,  since  the  eyes  under  such  circum- 
stances tend  instinctively  to  turn  their  axes  in  a  converging  di- 
rection towards  the  object  of 
vision.  At  least,  these  seem 
to  be  the  causes  of  failure  to 
the  writer,  who  has  endeavored 
perse veringly  at  various  times 
since  the  introduction  of  this 
binocular  to  overcome  the  diffi- 
culties of  its  use,  but  always 
with  such  loss  of  comfort  as 'to 
lead  to  an  abandonment  of  the 
attempt.] 

["Lately,  Mr.  Stephenson  of 
London  has  devised  a  binocular 
in  which  the  pencil  of  light  is 
bisected  by  a  pair  of  small 
prisms  inserted  very  close  to 
the  objective,  the  image  being 
erected  and  reflected  obliquely 
forward  by  a  mirror  or  prism 
placed  in  the  tube  above.  This 
arrangement  requires  the  stage 
to  be  permanently  fixed  in  a 
horizontal  position,  the  di- 
verging tubes  thus  taking  an 
inclined  position  favorable  for 
every  use.  It  is  not  converti- 
ble to  a  monocular.  Being  an 
erecting  binocular  it  is  especial- 
ly adapted  to  use  as  a  dissecting 
or  preparing  microscope.] 

[Meanwhile,  Mr.  F.  H. 
TTenham  also  of  London  had 
abandoned  the  idea  of  a  sym- 
metrical division  of  the  rays, 

and  introduced  a  little  prism  into  the  pencil  just  above  the  ob- 
jective, which  should  reflect  the  right  half  of  the  pencil  obliquely 


THE  BINOCULAR  MICROSCOPE.  41 

across  the  left  half  to  the  left  eye,  while  the  left  half  passed 
without  interruption  to  the  right  eye,  as  shown  in  section,  Fig. 
16,  where  A  is  the  "Wenham"  prism  which  reflects  half  the  light 
through  the  oblique  body  B,  and  D,  E  are  two  draw-tubes  by 
slightly  raising  or  lowering  which,  the  distance  apart  of  the  two 
eye  lenses  may  be  adapted  to  the  eyes  of  various  observers. 
Such  a  binocular  is  shown,  complete,  in  Plate  VII.  This  ar- 
rangement gives  one  field  of  vision  with  definition  absolutely 
unimpaired,  an  advantage  as  yet  gained  by  no  other  binocular. 
It  is  also  interchangeable  while  in  use  from  the  binocular  to  the 
monocular  effect,  by  simply  slipping  the  prism  into  or  out  of  the 
pencil  of  light.  These  advantages  proved  greatly  to  outweigh  its 
somewhat  cumbersome  mounting  which  is  not  removable  from  the 
stand,  the  unequal  light  and  definition  of  the  two  fields,  and  its 
practical  limitation  to  medium  powers  and  apertures ;  and  it 
immediately  became  and  has  thus  far  remained  the  binocular  of 
England  and  this  country.  It  is  best  adapted  to  powers  of 
from  one  to  two  inches  (25  to  51  mm.)  up  to  one-half  or  one- 
fourth  inch  (13  to  6  mm.).  With  low  powers  of  ample  aper- 
ture the  capacity  of  the  objective  is  sensibly  reduced  by  the 
brass  mounting  of  the  prism,  which  serves  as  a  diaphragm  to 
cut  down  this  aperture,  and  with  powers  of  from  one-half  inch 
(13  mm.)  upward,  the  angle  of  aperture  of  the  objective  used 
should  be  moderate,  and  the  illumination  should  be  arranged 
to  light  both  fields  freely  without  flooding  the  object  with  two 
much  light.] 

[Nachet  soon  changed  his  binocular  so  as  to  embody  some  of 
the  advantages  of  the  Wenham  form,  likewise  incorporated  into 
the  stand  itself,  allowing  one  portion  of  the  pencil  to  pass  with- 
out intentional  alteration  through  glass  with  parallel  surfaces 
directly  to  one  ocular,  while  the  other  portion  was  diverted 
twice  by  internal  reflection  in  two  prisms,  and  then  directed  ob- 
liquely to  the  other  ocular.  This  form  was  well  adapted  to  the 
small  stands  of  continental  model,  and  is  still  in  use.] 

[Just  now  another  binocular,  in  the  form  of  a  removable  ocu- 
lar, is  coming  into  use,  devised  by  Prof.  Abbe  and  manu- 
fractured  by  Zeiss  of  Jena.  In  arrangement  of  the  parts  and 
direction  of  the  rays  it  resembles  the  last  mentioned  form,  but 


42  THE  MICROSCOPE  IN  BOTANY. 

instead  of  a  division  of  the  light  into  lateral  halves  a  part  of  the 
light  from  the  whole  pencil  is  transmitted  to  one  eye,  and  the 
other  part  reflected  to  the  other.  By  stops  over  the  eye  lenses, 
cutting  off  the  opposite  lateral  portions  of  each,  the  fields  can 
be  so  differentiated  as  to  produce  a  stereoscopic  effect  when  de- 
sired.] 

[Of  the  numerous  ingenious  and  plausible  binoculars  invented, 
the  above  mentioned  are  all  which  have  come  into  actual  use 
sufficiently  to  demand  notice.  It  is  evident  that  the  binocular 
has  been  received  with  more  favor  here  and  in  England  than  on 
the  continent,  for  the  reason  that  it  is  better  adapted  to  the  work 
of  the  one  than  to  that  of  the  other.  It  is  most  applicable  to 
medium  powers  and  low  angles,  and  is  most  valued  by  those 
who  use  such  powers  for  general  natural  history  work,  where 
stereoscopic  effect  is  available  and  serviceable.  On  the  other 
hand,  it  is  least  valued  or  used,  if  at  all,  by  histologists,  medi- 
cal microscopistSjdiatomists,  and  other  classes  who  use  mostly  the 
higher  powers  and  larger  angles,  that  render  its  use  less  satis- 
factory if  not  inexpedient. — R.  H.  W.] 

[Y.  THE  EYE  SHADE.] 

[When  the  monocular  instrument  is  used,  the  fatigue  of  the 
observer's  eyes  is  greatly  lessened  by  habitually  keeping  the 
unemployed  eye  open,  and  protecting  it  by  a  black  eye-shade, 
placed  before  it.  The  greatest  comfort  is  attained  when  light 
is  arrested  by  a  central  stop  of  limited  size,  which  does  not 
wholly  darken  the  eye,  but  only  prevents  the  formation  in  it  of  an 
image  of  the  objects  on  the  table.  This  prevents  the  confusing 
effect  of  an  image  in  the  unused  eye,  and  the  fatiguing  effort 
required  to  keep  the  observer's  attention  confined  exclusively 
to  the  microscopic  image  in  the  other;  but  still  avoids  tiresome 
contrast  between  the  two  eyes  by  allowing  the  entrance  of  much 
diffuse  light,  and  permits,  without  abrupt  transfer  from  dark- 
ness to  light,  the  frequent  changes  required  from  rest  over  the 
shade  to  inspection  of  books  or  of  other  objects  upon  the  well- 
lighted  table.  For  this  reason,  probably,  and  for  their  own 
clumsiness,  the  large  shades  or  hoods  first  used  were  soon  aban- 


THE  EYE  SHADE.  43 

donecl.  Lately,  some  very  neat  and  useful  shades  have  been 
used  ;  but  being  attached  to  the  top  of  the  ocular,  they  could  be 
used  only  on  oculars  of  exactly  similar  size  and  form,  and  were 
invariably  removed  and  required  to  be  replaced  with  every 
change  of  ocular.  For  these  reasons  the  writer  caused  to  be 
constructed  a  new  form  represented  in  Fig.  17,  which  springs 
upon  the  compound  body  just  below  the  ocular,  and  without  re- 
gard to  the  style  of  ocular  or  fiuish  of  tube,  is  securely  held  at 


a  convenient  distance  from  the  face,  is  not  affected  by  change  of 
oculars,  and  can  be  instantly  transferred  to  any  microscope  body 
of  suitable  size,  or  reversed  to  shade  the  other  eye.  Being 
made  of  hard  rubber  it  is  of  proper  color  and  light  weight,  is 
little  inclined  to  scratch  the  brass  work  with  which  it  comes  in 
contact,  and  is  sufficiently  elastic  to  be  placed  without  altera- 
tion upon  tubes  varying  6  mm.  in  diameter.  It  is  made  by 
the  Bausch  and  Lomb  Optical  Co.,  of  Rochester,  of  any  size 
desired,  and  is  cheap  as  well  as  efficient.  The  writer's  expe- 
rience leads  him  to  believe  that  some  such  contrivance  should 
always  remain  fixed  upon  any  minocular  microscope  while  it 
stands  upon  the  table  ready  for  use,  even  occasional  glances 
into  the  tube  being  more  tiresome  without  this  accessory  than 
with  it.— R.  H.  W.] 

VI.     THE  MAGNIFYING  POWER  OF  THE  MODERN 
MICROSCOPE. 

The  optical  powers  of  a  microscope  depend  on  several  differ- 
ent factors.  A  microscope  which  will  satisfy  modern  require- 
ments must  give  a  large,  flat  and  bright  field  of  vision,  in  which 
the  magnification  of  the  margin  is  essentially  the  same  as  that 
of  the  middle.  The  image  produced  must  be  perfectly  distinct 
on  the  edges.  The  fine  structural  relations  of  the  object  must 


44  THE   MICROSCOPE   IX  BOTANY. 

be  sharply  brought  out,  and  the  magnifying  power  of  the  in- 
strument should  not  be  too  small.  The  size  and  brightness  of 
the  field  of  vision  depend  chiefly  upon  the  angle  of  aperture 
of  the  objective,  and  on  the  proper  position  of  the  field  lens  of 
the  ocular,  while  the  sharpness  and  colorlessness  of  the  image  are 
dependent  upon  the  correction  of  the  spherical  and  chromatic 
aberration. 

Under  the  designation  "resolving  power,"  we  understand  the 
capacity  of  the  objective  to  bring  to  view  the  fine  structural  rela- 
tions of  the  object ;  the  more  and  finer  these  relations  which  an 
objective  will  discover,  the  greater  is  its  resolving  power. 

Independent  of  this  quality  is  the  magnifying  power  of  the 
microscope,  that  is,  the  capacity  to  produce  an  image  of  an  ob- 
ject which  exceeds  in  extension  many-fold  the  object  itself.  In 
microscopical  magnification  we  speak  only  of  linear  enlarge- 
ment. It  is  expressed  in  terms  of  one  dimension  of  space. 
There  are  various  contrivances  and  methods  to  determine 
the  magnifications  of  a  microscope;  they  are  all,  however, 
grounded  upon  the  use  of  a  very  fine  measuring  scale  (  e.  #.,  a 
millimeter  divided  into  100  parts),  which  is  made  on  a  glass 
slip  and  viewed  urrder  the  microscope  to  determine  the  distances 
apart  of  two  or  more  division  marks  of  the  millimeter,  so  as  to 
find  thence  by  simple  division  the  amount  of  the  magnification. 
Latterly  the  opticians  very  exactly  determine  the  magnification 
of  the  various  combinations  of  their  lenses  and  furnish  a  table 
of  the  same  with  their  microscopes,  so  the  microscopist  is  now 
seldom  required  to  determine  for  himself  the  magnification  of 
an  object ;  for  this  reason  he  is  referred  to  the  more  compre- 
hensive works  of  other  authors.29 

The  magnification  is  produced  in  great  part  by  the  objective 
and  in  a  less  degree  by  the  ocular.  Each  objective  will  show 
different  magnifications  by  the  use  of  oculars  of  different  powers, 
since  the  image  produced  by  the  object  will  be  more  powerfully 
magnified  by  oculars  of  greater  curvature  than  by  those  of 
less. 

29  See  Harting,  1.  c.,  p.  131,  p.  244jf.;  Harting,  Recherches  Micrometriques— II.  v.  Mohl, 
1.  c.,  p.  215jT.— Jacquin  in  Zeitsch.  /.  Physik  &  Mathcmatik.  18-28  [IV],  p.  1.— Ettingliausen 
ibid  1829  (V),  p.  316jf.— Pohl  in  Berichte  d.  k.  k.  Acad.  d.  Wiss.  Wien,  XI,  p.  504J.— Dip- 
pel,  1.  c.,  p.  92-100,  etc. 


THE  MAGNIFYING  POWER. 


45 


In  order  to  show  in  what  relation  these  different  glasses  some- 

o 

times  stand,  we  will  take  an  example  from  the  magnifications 
which  the  Hartnack  oculars  1-6  give  in  combination  with  his 
objectives  No.  2,  4,  5,  7,  10. 


OCULARS. 

OBJECTIVES. 

1 

2 

3 

4 

5 

6 

2 

25 

30 

45 

4 

60 

70 

90 

140 

5 

100 

125 

160 

240 

7 

200 

240 

300 

450 

600 

750 

10 

520 

600 

750 

1100 

1500 

1800 

It  is  not  a  matter  of  indifference  in  what  manner  the  oculars 
are  combined  with  the  objectives.  The  principal  burden  should 
always  be  laid  upon  the  objective.  For  the  production  of  a 
given  magnification,  there  should  be  combined  the  strongest 
possible  objective  with  the  weakest  possible  ocular.  Resolving 
power,  the  delineation  of  the  details  of  the  image,  is  alone  an 
attribute  of  the  objective.  The  ocular  does  indeed  more  or  less 
enlarge  the  image  produced  by  the  objective,  and  indeed,  nat- 
urally at  the  cost  of  its  brightness,  but  it  will  have  only  the  least 
possible  influence  upon  its  improvement,  on  the  resolution  of 
the  finer  structural  relations  of  the  image.  Supposing  we  de- 
sired to  produce  a  magnification  of,  say,  240  diameters,  in  ac- 
cordance with  the  above  quoted  table,  we  should  not  combine 
ocular  4  with  objective  5,  but  objective  7  with  ocular  2.  Or  if 
we  desired  a  magnification  of  600  diameters,  we  should  not 
take  objective  7  and  ocular  5,  but  objective  10  and  ocular  2. 
Or  if  we  wanted  750  diameters,  we  should  not  put  objective  7 
with  ocular  6,  but  objective  10  with  ocular  3. 

The  amount  of  linear  magnification  attainable  to-day  with  a 
good  microscope  is  a  very  considerable  one,  especially  is  that 
of  the  immersion- systems  abreast  of  what  formerly  could  be 
produced  with  dry  lenses,  and  was  called  a  very  prodigious  one. 
In  order  to  give  an  idea  of  what  this  really  is,  we  will  instance 
the  objective  mngnifications  of  instruments  from  some  of  our  best 


46 


THE   MICROSCOPE   IN   BOTANY. 


optical  manufactories.  In  each  case,  only  the  linear  magnifi- 
cation will  be  given  which  would  be  produced  by  the  use  of  the 
weakest  ocular  which  the  firm  furnishes.  An  asterisk  (*)  indi- 
cates that  the  magnification  is  produced  by  an  immersion-system, 
the  rest  are  by  dry  lenses. 

TABLE  OF  MAGNIFICATIONS. 


MAKERS. 

OCULARS. 

OBJECTIVES  AND  MAGNIFICATIONS. 

No.  0     1      2 

34567          8          9 

10         11 

Nachet. 

No.  1. 

30,  89,  180, 

2GO,  300,  350,  460,*  580,*  775,*  900,* 

1150,*  1320, 

12 

1700.* 

No.  1      2      3 

45678           9            10 

11        12 

Hartnack. 

No.  1. 

15,  25,  50, 

60,  100,  150,  200,  250,  350,  410,*  520 

,*  600,*  710,* 

13      14 

15         16          17          18 

820,*  930,* 

1040,*  1200,*  1400,*  1560.* 

No.  9  is  both  dry  and  immersion. 

No.  00     0      I 

II     III       IV        V       VI      VII      VIII 

IX            X 

Seibert. 

No.O. 

10,    18,    30, 

45,    66,    100,   200,  305,  460,*  650,* 

950,*    1450.* 

No.  123 

4567         8         9        10 

11         12 

Schieck. 

No.  0. 

20,    40,    70 

90,    150,    200,    275,    400,    450,    500,*    600,*   750,* 

13         14 

15           16         • 

850,*    930,*    1100,*    1400.* 

No.  a    aa    A 

AA     B       BB       C          CC         U       DD 

E 

Zeiss. 

No.  1. 

5,    18,  45, 

70,    110,    180,    220,*   240,    380,    400,* 

680.* 

No.  1      2      3 

4        5         6        7         8         9        10 

11 

Winkel. 

No.  1. 

25,    54,    74 

102,    184,    222,    275,    366,    458,    500,    584. 

In  the  following  table  is  presented  the  strongest  ocular  mag- 
nification of  the  above  named  six  firms. 


MAKER. 

OBJECTIVE  SYSTEM. 

OCULAR. 

MAGNIFICATION. 

Schieck. 

System  15. 

No.  5. 

6500*  Diameters. 

Hartnack. 

"      18. 

No.  6. 

5400*             " 

Nachet. 

"       12. 

No.  4. 

4500*             '< 

Seibert. 

"       X. 

No.  III. 

4400*             " 

Zeiss. 

Imm.      "        3. 

No.  5. 

2300*            '  " 

Winkel. 

"      11. 

No.  6. 

1600               « 

THE  MAGNIFYING  POWER. 


47 


[In  America  and  England,  objectives  are  universally  named 
from  their  equivalent  focal  lengths  in  inches  ;  a  one-inch  object- 
ive, for  instance,  being  a  lens  system  whose  amplifying  power 
is -the  same  as  that  of  the  simple  lens  of  the  specified  focal  dis- 
tance. This  nominal  focal  distance  is  usually  much  greater  than 
that  called  the  working  focal  distance,  between  the  object  and 
the  nearest  portion  of  the  combination  of  lenses.  Oculars  are 
similarly  named  by  several  makers.  It  follows  from  this  most 
convenient  system  of  nomenclature,  that  the  amplification  is 
approximately  made  known  by  the  name.  Thus  assuming,  ac- 
cording to  usage,  the  round  number  10  inches  (25  cm.)  to  be 
the  distance  of  most  distinct  unaided  vision,  a  one  inch  (25 
mm.)  lens  would  magnify  ten  times,  a  two  inch  (51  mm.)  five 
times,  a  one-half  inch  (13  mm.)  twenty  times,  a  one-fifth  inch, 
(5  mm.)  fifty  times,  etc.  Furthermore,  the  combined  amplifi- 
cation of  objective  and  ocular,  as  used  in  the  compound  micros- 
cope at  a  distance  of  ten  inches  (25  cm.)  from  each  other,  will 
be  represented  by  the  multiple  of  their  individual  powers  ;  a 
one-fourth  objective,  for  instance  (  X  40)  with  a  one-inch  ocular 
(  X  10)  giving  a  resultant  power  of  400.  The  following  table 
shows  the  theoretical  powers,  thus  calculated,  of  a  few  combi- 
nations, other  powers  being  in  the  same  proportion  : 


OCULARS. 

OBJECTIVES. 

1 

1 

1 

1 

1 

1 

1 

« 

^ 

5 

3 

1 

2 

5 

10 

15 

20 

30 

50 

*c   ^ 

2 

o  S 

0    § 

£ 

127 

76 

25 

13 

5 

2.5 

1.7 

1.3 

0.8 

0.5 

*  3 

£ 

POWER. 

2 

4 

10 

20 

50 

100 

150 

200 

300 

500 

In. 

mm. 

a 

2 

51 

5 

§ 

10 

20 

50 

100 

250 

500 

750 

1000 

1500 

2500 

1 

25 

10 

*2   H 

20 

40 

100 

200 

500 

1000 

1500 

2000 

3000 

5000 

| 

13 

20 

I  1 

40 

80 

200 

400 

1000 

2000 

3000 

4000 

6000 

10000 

* 

6 

40 

z 

80 

160 

400 

800 

2000 

4000 

6000 

8000 

12000 

20000 

[Such  tables  have  been  used  for  an  indefinite  period  as  a 
means  of  assisting  the  memory  in  respect  to  the  powers  em- 
ployed. They  have  not  been  used  with  the  understanding  that 


48 


THE   MICROSCOPE   IN   BOTANY. 


they  were  precisely  accurate  as  applied  in  practice,  but  with  the 
distinct  explanation  that  they  were  only  approximately  correct, 
since  it  would  be  required,  to  make  them  literally  correct,  to 
locate  the  ocular  and  objective  by  means  of  certain  optical 
planes  which  obviously  do  not  coincide  with  any  recognizable 
part  of  the  mounting,  and  which  cannot  be  determined,  by  any 
known  method,  with  sufficient  facility  for  popular  use.  It  has, 
therefore,  been  somewhat  customary  to  approximate,  by  taking 
an  eight  and  one-half  inch  (21  cm.)  length  of  tube,  assuming 
that  the  rest  of  the  ten  inches  (25  cm.)  will  be  more^  or  less 
evenly  supplied  by  the  mounting  of  the  objective  and  ocular. 
The  position  of  the  planes  certainly  vary  much  in  different  lens 
systems  of  equal  power,  but  they  are  stated  to  be  usually,  near 
the  diaphragm  of  the  ocular,  but  more  or  less  behind  the  back 
lens  of  the  objective.  This  explains  the  over-estimate  of  am- 
plifications, especially  in  the  lower  powers,  obtained  by  com- 
putation. It  also  shows  that  exact  accuracy  in  such  estimates 
is  scarcely  to  be  expected  under  any  plan  that  may  be  adopted, 
and  that  to  attain  even  average  accuracy,  it  would  be  necessary 
either  to  lengthen  the  tube  to  an  inconvenient  extent,  or  else  to 
habitually  underrate,  by  general  agreement,  the  powers  of 
either  the  oculars  or  the  objectives.] 

[The  following  table  represents  the  magnifiying  powers  of 
the  Bausch  and  Lomb  objectives  and  oculars  in  their  various 
combinations,  determined  by  actual  measurement  with  a  mi- 
croscope tube-length  of  eight  and  one-half  inches  (21  cm.)  in 
addition  to  the  objective  and  ocular.] 


S" 

OBJECTIVES. 

J 

3 

1 

4 

1 

j 

1 

1 

1 

§ 

4 

3 

2 

1 

1 

To 

4 

5~ 

IT 

8 

10 

12 

16 

A 

a 
c 

12 

18 

25 

4B 

50 

92 

130 

210 

275 

325 

400 

550 

650 

800 

B 

o 

15 

23 

30 

54 

70 

110 

160 

250 

325 

390 

490 

650 

775 

9SO 

C 

ft 

23 

30 

45 

80 

90 

165 

240 

375 

485 

580 

750 

970 

1160 

1500 

D 

£ 

30 

45 

60 

108 

140 

220 

320 

500 

650 

780 

980 

1300 

1550 

1960 

^ 

1 

TABLE  OF  OBJECTIVES.  49 

[By  including  in  this  table,  objectives  up  to  one-fiftieth,  and 
oculars  up  to  one-fourth  or  one-eighth,  all  of  which  are  made, 
the  resultant  powers  would  extend  well  up  into  the  tens  of 
thousands ;  such  powers,  however,  are  but  little  used  or  es- 
teemed. These  oculars  do  not  correspond  to  even  fractions  of 
an  inch,  and  are  here  designated  by  letters,  after  the  early  Eng- 
lish style  which  is  not  yet  wholly  obsolete  ;  but  the  makers  in- 
tend hereafter  to  conform  to  the  numerical  plan.] 

[As  the  nominal  focal  lengths  of  objectives,  while  liable  to 
moderate  errors  and  discrepancies,  indicate  the  amplifying 
powers  sufficiently  well  for  most  purposes,  it  is  only  necessary 
in  stating  the  powers  made  by  the  American  manufacturers,  to 
tabulate  the  equivalent  focal  lengths  of  their  objectives.  As 
to  oculars,  the  lowest  is  usually  a  one  and  one-half  or  two  inch 
(38  to  51  mm.)  ;  the  one  inch  (25  mm.)  is  much  used  and  is 
said  to  be  the  power  employed  by  prominent  makers  in  correct- 
ing their  objectives,  and  those  of  from  one-half  inch  (13  mm.) 
upwards  are  used  for  such  special  purposes  as  inicrornetry,  or 
counting  fine  lines.] 

[The  amplifications  with  two  inch  (5cm.)  ocular,  given  in  the 
fourth  column  of  the  table  on  pages  50  and  51,  are  calculated 
for  the  ten  inch  (25  cm.)  distance,  and  are  therefore  somewhat 
overrated  practically.  It  should  be  added  that  the  highest  powers 
named  are  seldom  if  ever  made  or  used ;  and  that,  to  say  the 
least,  the  work  of  most  microscopists  can  be  done  more  easily, 
if  not  better,  with  a  1  or  JL  than  with  a .  JL  or  A.] 

[In  the  table  the  latest  published  price  (1884)  of  each  lens 
is  annexed,  to  show  the  present  value  of  such  work,  and  to  in- 
dicate the  significant  relation,  in  the  various  lenses  of  each 
maker,  between  angular  aperture  and  price.  In  a  few  instances 
other  considerations  overrule  this  law,  and  give  an  unexpectedly 
high  or  low  figure.  It  is  claimed  by  the  makers,  though  often 
earnestly  disputed  by  others,  that  the  excessive  prices  of  the 
high-angled  lenses  are  not  fictitious  values  based  upon  a  monop- 
oly of  the  supply,  but  they  only  adequately  represent  the 
amount  of  labor  actually  expended  in  overcoming  the  practical 
difficulties  of  construction.  On  the  other  hand,  it  is  certain 
that  the  day  has  fortunately  passed  away  when  lowness  of  price 
4 


50 


THE   MICROSCOPE   IN  BOTANY. 


TABLE  OF  AMERICAN  OBJECTIVES. 


roc 

8 

1 

US. 

c 

£ 

POW 

« 

B 

® 

<1 

ER. 

^  J5 
P 

BAUS 
LOJ 

9 

5 
f 

o 
Pi 

<;  ° 

CH  & 

IB. 

1 

3 

$ 

Aperture  o 

ft 

°  cs 

•ow. 

oo 
$ 

Aperture  § 

X 
0 

.ACH. 

to 

6 
$ 

Aperture  £g 
0  V. 

JER. 

1 

3 

Is 

Aperture  ^ 

0  > 

r,ES. 

1o 

O 

3 
$ 

ZEN 
MAYI 

£  - 

<  ° 

T- 

:R. 

"a 
$ 

5 

127 

2 

10 

/5-3\ 
UnJ 

18 

/5-3\ 
\in.) 

20 

(**} 
\ln./ 

15 

4 

102 

24 

13 

6 

10 
12 

6 

13 
18 

12 

18 

8 
/4-2\ 
UnJ 

8 
20 

1 
/4-2\ 

VtaJ 

8 
20 

9 

12 

3 

76 

8* 

17 

9 
12 
16 

6 
13 

18 

12 

18 

11 
13 

8 
20 

8 
13 

7 
20 

12 

17 



2 

4 

1 

51 

5 
61 

25 
33 

12 
15 
22 

6 
13 
18 

15 
20 
22 

15 
J8 
20 

15 

18 

6 
20 

10 
16 
20 

7 
18 
30 

38 

16 
24 
32 

6 
15 

20 

15 
22 
30 

12 

18 
25 

18 
24 

6 
18 

23 

17 

23 

15 

25 

10 

50 

20 

36 
45 

6 

15 
25 

25 
30 
50 

15 
20 
35 

15 
24 
26 
36 

85 

5 
12 
25 

20 
80 

25 
33 
40 

12 

22 
40 

25 

17 

8 
10 

3 

4 

20 
19 

12 

62 









26 
?2 

10 

18 

13 

67 

27 
40 

8 
15 

2 
3 

17 

15 
20 

75 

100 

25 
35 

65 

15 
22 
30 

25 

36 
40 
43 

6 

10 
15 
25 

32 
86 

47 

12 
22 

30 

30 

17 

32 

18 

1 
2 

13 

42 

65 

98 

9 
18 

30 

65 

18 

27 

40 
50 

72 
110 

7 

10 
20 

40 
50 

50 
n 
70 
100 

15 

25 

50 

4 
10 

1 
3 

10 

25 
30 

125 

150 

70 
110 

13 
34 

50 

75 
100 

18 

25 
35 

60 

80 
110 

20 

20 

to 



75 

95 
115 

30 

35 
40 

60 
80 

22 
30 

8 

120 

35 

1 
4 

1 
5 

6 
5 

40 
50 

200 
250 

n 
75 

100 
125 

na.h 
1.40 

14 
17 

24 

100 

n 
75 

20 

n 
75 
n 

80 

na.hi 
1.20 

14 
25 

60 

n 
100 

115 
13 
na.di 
1.28 

16 

30 

40 

70 

100 

135 

i 
170 

25 

90 
120 

18 
35 

110 
130 

18 
28 

90 
110 

135 

20 
35 
40 

135 
na.hi 
1.40 

22 

90 

35 
40 

THE  OBJECTIVE-SYSTEM. 


51 


TABLE  OF  AMERICAN  OBJECTIVES.— Continued. 


Inches  3  1 

us. 

i 
s 

PO^ 

\Viih2in.  w 
ocular  F 

fS 

«  a.mi.iodv 

CH& 
MB. 

•3 
o 

i 

Aperture  O 

*  d 

ow. 

6 
$ 

GUXD 

£ 

!o 

LACH. 
* 

Aperture  " 

w 

«  V, 
rt 

ER. 

5 
1 

Aperture  ^ 

LES. 

1 

$ 

ZEN 
MAY 

0 

1 
s. 

T- 

KR. 

« 

,0 

$ 

1 
6 

4 

60 

300 

140 

i 
165 
na.hi 
1.18 
na.hi 
1.40 

40 
23 
45 
70 

n 

100 

110 
165 

20 
25 
40 

n 

80 
na.gi 
1.12 
na.hi 
1.20 

16 
30 
45 

di 

175 
na.i 
1.21 

na.lii 
1.35 

40 
40 
70 

1 
b 

3 

80 

400 

115 

135 
i 
170 
na.hi 
1.18 
na.hi 
1.40 

21 
30 

25 
50 
75 

150 

45 

135 

na.hi 
1.40 

24 

80 

120 
i 
135 
di 
135 

150 
di 
175 

25 
25 
32 
45 

40 

1 
10 

1 
12 

2.5 
2.1 

100 
120 

500 

170 
na.hi 
1.18 
na.hi 
1.40 

28 
50 
80 

130 

i 
175 

30 
50 

na.gi 
1.12 
na.hi 
1.20 

25 
35 

na.i 
1.25 
na.hi 
1.27 
nahi 
1.35 

50 
60 

80 

135 
170 

35 
45 



600 

130 

i 
175 
na.hi 
1.18 
na.hi 
1.40 

27 

30 
55 
90 

135 

na.hi 
1.40 

40 
90 

1 
15 

1.7 

150 

750 

i 
160 

75 

i 
150 

150 

na.i 
1.17 

40 

60 
66 

i 
170 

65 

1 
16 

1 
18 

1.6 

160 
180 

.800 

i 
175 
na.hi 
1.18 
na.hi 
1.40 

35 
65 
125 

na.gi 
1.12 
na.hi 
1.20 
na.hi 
1.40 

40 
50 
120 

135 

na.hi 
1.35 

40 
125 



— 



1.4 

900 

di 
175 

70 

1 
20 

1 
25 

1.3 

200 

1000 

i 
170 

100 

na.hi 
1.20 
na.hi 
1.40 

75 

160 

160 

1 

250 

1250 

na.hi 
1.40 

200 

na.hi 
1.20 
nahi 
1.40 

120 
220 

na.hi 
1.35 

150 

100 

1 
32 

1 
50 

0.8 

320 

1600 

na.hi 
1.20 

150 



0.5 

500 

2500 

na.hi 
1.20 

200 

na.hi 
1.17 

270 

1 
75 

0.3 
0.2 

750 

3750 
9901 

na.hi 
1.20 

260 

na.lii 
1.17 

??? 

1 
100 

1000 

na.hi 
1.20 

300 

[References  in  table:  i  =  immersion;  di  =  dry  or  immersion  ;  gi  =  glycerine  immersion ; 
hi  =  homogeneous  immersion;  na=numerical  aperture;  n  =  non-adjustable,  all  lenses 
of  over  65°,  if  not  so  marked,  having  screw  collar  adjustment  for  thickness  of  cover.] 


52  THE   MICKOSCOPE   IN   BOTANY. 

indicated  poorly  finished  or  imperfectly  corrected  lenses ;  the 
cheapest  objectives,  by  the  best  makers,  being  carefully 
corrected  and  neatly  mounted,  and  owing  their  cheapness  to  the 
ease  with  which  low-angled  lenses  can  be  corrected  and  the 
simplicity  with  which  they  may  be  mounted.  They  are  there- 
fore adequate  for  any  work,  however  important,  that  requires 
the  use  of  such  angles ;  and  they  are  most  commonly  supplied 
in  connection  with  the  various  grades  of  students'  and  physi- 
cians' microscopes.] 

[The  focal  distances  and  angular  apertures  in  the  table  on 
opposite  page  are  given  on  the  authority  of  the  several  makers. 
There  is  reason  to  believe  that  these  data,  on  account  of  ex- 
tensive agitation  of  the  subject,  are  more  carefully  determined 
and  more  accurately  stated  than  formerly.  The  apertures  given 
are  the  air-angles,  unless  otherwise  stated.  In  lenses  of  the 
highest  grade  the  numerical  aperture  is  necessarily  given,  there 
being  no  corresponding  air-angle.  The  Aperture  Table  of  the 
Royal  Microscopical  Society,  founded  upon  the  elaborate  re- 
searches of  Prof.  Abbe,  is  reproduced  here  as  a  most  convenient 
means  of  comparing  dry,  water  and  homogeneous  immersion 
lenses,  and  of  applying  the  doctrine  of  Numerical  Aperture  as  a 
measure  of  their  theoretical  illuminating,  resolving  and  penetrat- 
ing powers.  For  instance,  180°,  the  theoretical  maximum  of  air 
angle,  corresponds  to  unity  of  numerical  aperture,  and  of  illu- 
minating and  penetrating  power ;  this  being  practically  equiva- 
lent to  only  97°  31'  of  water  angle  or  82017'  in  homogeneous 
media  corresponding  to  crown  glass,  and  having  a  theoretical 
resolving  capacity  for  96,400  lines  to  the  English  inch.  Higher 
apertures,  as  readily  shown  by  the  table,  are  practicable  only 
in  immersion  lenses,  and  give  increased  illuminating  and  resolv- 
ing but  lessened  penetrating  powers ;  lower  apertures  having 
exactly  the  reverse  characteristics.  R.  H.  W.] 

There  is  a  widespread  opinion  that  the  more  a  microscope 
magnifies  the  better  it  is.  This  is  really  the  case,  however, 
only  under  certain  conditions.  A  microscope  which  magni- 
fies very  powerfully  but  at  the  same  time  gives  imperfect, 
indistinct,  and  poorly  illuminated  images  is  far  less  to  be  preferred 
than  one  which  gives  much  smaller  magnification  but  produces 


APEKTITKE    TABLE. 


The 


"  APERTURE"  of  an  optical  instrument  indicates  its  greater  or  less  capacity  for  re- 
ceiving rays  from  thu  object  and  transmitting  them  to  the  image,  and  the  aperture  of  a 
Microscope  objective  is  therefore  determined  by  the  ratio  between  its  local  length  and 
the  diameter  of  the  emergent  pencil  at  the  plane  of  its  emergence  —  that  is,  the  utilized 
diameter  of  a  single-lens  objective  or  of  the  back  lens  of  a  compound  objective. 
This  ratio  is  expressed  for  all  media  ami  in  all  cases  by  n  sin  u,  n  being  the  refractive  index 
of  the  medium  and  u  the  semi-angle  of  aperture.  The  value  of  n  sin  u  for  any  partic- 
ular case  is  the  "numerical  aperture"  of  the  objective. 


Diameters  of  the 
back  lenses  of  various 
Dry  and  Immersion 
objectives  of  the  same 
power  (4  in.) 
from  0-50  to  1  52  N.  A 

Numerical 
Aperture. 
(7t  bin  u  =  a. 

Angle  of  Aperture  (=  2  u.} 

Illuminating 
power. 

(«2-) 

Theoretical 
resolving 
power,  in 
lines  10  an  Inch. 
(A  =  0-S2C9  M 
=  line  K.I 

Penetrating  power. 
(^) 

if! 

"o 

Water- 
iwmerKion 
objectives. 
(?t  =  1-33) 

Ilovwf/eneons- 
immcrxion 
olljcctives. 
(«  =  l-52.) 

1-52 

.. 

180°    0' 

2-310 

140.528 

•658 

1-50 

.. 

.. 

161°  23' 

2-250 

144,000 

•607 

1-48 

.. 

153°  39' 

2-190 

142,072 

•076 

1-46 

>t 

.. 

147°  42' 

2-132 

140,744 

•685 

1-44 

M 

.. 

142°  40' 

2-074 

138,816 

•094 

1-42 

.. 

138°  12' 

2-016 

136,888 

•704 

1-40 

134°  10' 

1-900 

134,900 

•714 

1.38 

>> 

tm 

130°  20' 

1-904 

133,032 

•725 

1-36 

rt 

126°  57' 

1  850 

131,104 

•735 

1-34 

.. 

123°  40' 

1-790 

129,170 

•746 

1-33 

tt 

180°    0 

122°    6' 

1-770 

128.212 

•752 

1-32 

105°  50 

120°  33' 

1-742 

127,248 

•758 

1-30 

M 

155°  38 

117°  34' 

1-0'JO 

125,320 

•709 

1-28 

148°  28 

114°  44' 

1-038 

123,392 

•781 

1-26 

142°  39 

111°  59' 

1-588 

121,464 

•794 

1-24 

137°  30 

109°  20' 

1-538 

119,536 

•806 



1-22 

133°    4 

106°  45' 

1-488 

117,608 

•820 

X^"~~^^^ 

1-20 

.. 

128°  55 

104°  15' 

1-440 

115,080 

•833 

ff            ^\\ 

1-18 

125°    3 

101°  50' 

1-392 

113,752 

•847 

M      1*16     j] 

1-16 

121°  20 

99°  29' 

1-340 

111,824 

•802 

v            JJ 

114 

1€ 

118°  00 

97°  11' 

1-300 

109,896 

•877 

^^^^s 

1-12 

lt 

114°  44' 

94°  50' 

1  254 

107.908 

•893 

1-10 

111°  30' 

92°  43' 

1-210 

100,040 

•909 

Q 

1-08 
1-06 
1-04 
1-02 
100 
0-98 

180°'    0' 
157°    2' 

108°  30 
105°  42' 
102°  53' 
100°  10' 
97°  31' 
94°'50' 

90°  33' 
88°  20' 
80°  21' 

84°  18' 
82°17'i 
80°  17' 

1-100 
1-124 
1-082 
1-040 
1-000 
•900 

104,112 
102,184 
100,250 
98,328 
90.400 
94,472 

•920 
•943 
•902 
•980 
1-000 
1-020 

0-96 

147°  29' 

92°  24' 

78°  20'!    -922 

92,544 

1-042 

0 

0-94 
O-92 
0-90 
0-88 

140°    6' 
133°  51' 
128°  19' 
123°  17' 

89°  50' 
87°  32' 
85°  10' 
82°  51' 

76°  24'     -884 
74°  30'     -840 
72°  30'     -810 
70°  44'     -774 

90,616 

88,088 
80,700 
84.832 

1-004 
1-087 
1-111 
1-136 

086 

118°  38' 

80°  34' 

68°  54'     -740 

82.904 

1-163 

0-84 

114°  17' 

78°  20' 

67°    0'      -700 

80,976 

1-190 

/*  s\ 

0-82 

110°  10' 

70°    8' 

05°  18';     -072 

79.048 

1-220 

(    *80  / 

0-80 

106°  10' 

73°  58' 

03°  31'     -040 

77.120 

1-250 

V       J 

0-78 

102°31' 

71°  49' 

01°  45'     -008 

75,192 

1-282 

^+~~*S 

0-76 

98°  50' 

09°  42' 

60°    0'     -578 

73.264 

1-316 

0-74 

95°  28' 

67°  30' 

58°  10'     -548 

71^336 

1-351 

0-72 

92°    (>' 

05°  32' 

56°  32'     -518 

09.408 

1-389 

0-70 

88°  51' 

63°  31' 

54°  50'     -490 

07,480 

1-429 

0-68 

85°  41' 

61°  30' 

53°    !)'     -462 

05,552 

1-471 

0-66 

82°  30' 

59°  30' 

51°  28'!    -430 

03,024 

1-515 

0-64 

79°  35' 

57°  31' 

49°  48'     -410 

01,096 

1-562 

0 

0-64 
0'60 
0-58 

70°  38' 
73°  44' 
70°  54' 

55°  34' 
53°  38' 
51°  42' 

48°    9'     -384 
40°  30'     -360 
44°  51'     -330 

59,708 
57,840 
55,912 

1-613 
1-667 
1-724 

0-56 

68°    6' 

49°  48' 

43°  14'     -314 

53,984 

1-786 

0  54 

65°  22' 

47°  54' 

41°  37'     -292 

52,050 

1.852 

0-52 

62°  40' 

46°    2' 

40°    0'     -270 

50,128 

1.923 

1 

0-50 

00°    0' 

44°  10' 

38°  24'     -250 

48,200 

2-COO 

EXAMPLE.— The  apertures  of  four  objectives,  two  of  Avhicli  are  dry.  one  water-immersion, 
and  one  oil-immersion,  would  be  compared  on  the  angular  aperture  view  as  follows: — 
10(>°  (air),  157°  (air),  142°  (water),  130°  (oil). 

Their  actual  apertures  are,  however,  as  -80  (air),  -98  (air),  1'26  (water),  1-38  (oil),  or  their 
numerical  apertures. 


COMPARISON  OF  ENGLISH  AND  METRIC  MEASURES. 


Scale    show- 
ing the 
rel;itioii  of 
Millimeters, 
etc.,  to 
Inches, 
mm. 
and 
cm.          in. 

Jfierc 

M         ins. 
1       -000039 
2      -000079 
3      -000118 
4      -000157 
5      -000197 
6      -000236 
7      -000276 
8      -000315 
9      -000354 
10      -000394 

11      -000433 
12      -OOU472 
13      -000512 
14      -00055  1 
15      -000591 
16      -000030 
17      -000669 
18       -00071,9 
19      -000748 
2O      -000787 

21      -000827 
22      -000866 
23    x  -000906 
24      -000945 
25      -000984 
26      -001024 
27      -001063 
28      -001102 
29      -001142 
30      -001181 

31      -001220 
32      -U01260 
33      -001299 
34      -001339 
35      -001378 
36      -001417 
37      -00  14.-)  7 
38      -001496 
39       -001535 
4O      -001575 

41      -001614 
42       -001654 
43      -001(593 
44      -001732 
45      -001772 
46      -001811 
47      -001850 
48      -001890 
49       -001929 
50      -001969 

60       -002302 
70       -002756 
80       -003  1  50 
90      -003543 
100      -003937 
200      -007874 
300      -011811 

millimeters,  etc.,  int 
mm.                     ins. 

1                     -039370 
2                    -078741 
3                 -118111 
4                   -157482 
5                     -190852 
6                    -236.'23 
7                    -275593 
8                    -314963 
9                    -354334 
10  (1  cm.)  -393704 
11                    -433075 
12                    -472445 
13                   -511816 
14                   -551186 
15                   -590550 
16                    -629927 
17                    -669297 
18                   -70*668 
19                   -748038 
2O  (2  cm.)    -787409 

21                    -826779 
22                   -866150 
23                   -905520 
24                   -944890 
25                     -984261 
Z6                  1-023631 
27                 1-063002 
28                 1-102372 
29                1-141743 
3O  (3  cm.)  1-181113 

31                 1-220483 
32                 1-259854 
33                   1-299224 
34                 1.338595 
35                  1-377965 
36                 1-417336 
37                 1-456706 
38                 1-496076 
39                 1-535447 
4O  (4cm.)  1-574817 

41                 1-614188 
42                 1-653558 
43                 1  -692929 
44                 1-732299 
45                 1-771669 
46                 1-811040 
47                 1-850410 
48                1-889781 
49                 1-929151 
5O  (5cm.)  1-968522 

decim. 

1 
2 
3 

4 
5 
6 
7 
8 
9 
10  (1  mete 

j  Inches,  etc. 
mm.                              ins. 
51                     2-007892 
52                    2047262 
53                     2-086633 
54                    2-126003 
55                    2-165374 
56                    2-204744 
57                   2-244115 
58                     2-283485 
59                     2322855 
60  (6  cin.)     2-362226 

61                     2-401596 
62                    2-440907 
63                     2-480337 
64                     2-519708 
65                     2-559078 
66                    2-598449 
67                    2-637819 
68                    2-677189 
69                    2-716560 
7O  (7  cm.)     2-755930 

71                      2-795301 
72                     2-834671 
73                     2-874042 
74                     2-913412 
75                      2-952762 
76                      2-992153 
77                     3-031523 
78                    3-070«94 
79                      3-110264 
80  (8  cm.)     3-149635 

81                     3-189005 
82                     3228375 
83                      3-267746 
84                     3-307116 
85                    3-340487 
86                    3-385857 
87                      3-425228 
88                      3-464598 
89                    3-503968 
9O  (9  cm.)     3-543339 
91                    3-582709 
92                    3622080 
93                    3-661450 
94                    3-70082U 
95                    3-740191 
96                     3-779561 
97                      3-818932 
98                    3-858302 
99                    3-897673 
100  (10  cm.  =1  dcm.) 

ins. 

3937043 
7-874086 
11-811130 
15-748173 
19-685216 
23-622259 
27-559302 
31-490346 
35-433389 
•)  39  370432 
=   3  280869  ft. 
=    1-093623  yds. 

Inches,  etc.,  into 
micro- 
millimeters,  etc. 
ins.            ft 
T^TTO      1-015991 
Tufoo       1-269989 
-ndnnr      1-693318 
To-tanr      2-539977 
snAnr       2-822197 
Tifoir       3-174972 
ToW       3-628539 
FoW       4-233295 
TuW       5-079954 
4iiW       6-349943 
snAnr       8-466591 
at-tar     12-699886 
-roW     25-399772 
mm. 
aio           -028222 
-8ta          -031750 
7^0-          -036285 
d-u           -042333 
•050300 
•056444 
•063499 
;rb          -072571 
ib          -084666 
^b          -101599 
-dro           -126999 
T5o-          -169332 
T£O           -253998 
5*6            -507995 
1-015991 
-2V         1-269989 
iV         1-587486 
-iV         1-693318 
i^         2-116648 
iV         2-539977 
i          3-174972 
4-233295 
&         4-762457 
|          5-079954 
(5-349943 
-^         7.937429 
|          9-524915 
cm. 
•fr          1-111240 
^          1-269989 
iV         1-428737 
*          1-587486 
U         1-740234 
1-904983 
fl|-         2-063732 
I          2-222480 
||         2  381229 
1          2-539977 
2          5-079954 
3          7-619932 
decim. 
4          1-015991 
_5          1-269989 
6          1-523986 
7          1-777984 
8          2.031982 
9           2-285979 
10          2  539977 
11           2-793975 
1  ft.        3-047973 
meters. 
1  yd.  =    -914392 

si 

ifl 

*; 

- 

; 

£    : 

- 

3 

E 

a  : 

- 

f 

"  = 

E 

«n  £ 

I 

_  to 

*    I 

L 

E 

to    - 

10    - 

*  C 

:• 

I 

•0   p 

oi  r 

I 

400      -015748 
500      -019685 
600      -023622 
700       -027559 
800       -U314!)(5 
900      -035433 
1000  (=1  mm.) 

1000  /u.  =  1  mm. 
10  mm.  =    cm. 
10  cm.  =  1  (in., 
10  dm.  =  1  m. 

TESTING  THE  OPTICAL  POWERS.  53 

clear  and  sharp  images.  It  may  be  said,  on  the  contrary,  that 
those  instruments  are  comparatively  the  best  which,  with  rela- 
tively low  magnifications,  still  show  the  details  which  appear  in 
poor  instruments  only  with  the  use  of  high  powers. 

In  judging  of  a  microscope  the  first  things  to  be  thought  of 
are  these :  Does  it  show  a  sharp  and  clear  image,  all  details, 
all  fine  structural  relations?  What  are  its  defining  and  resolv- 
ing powers?  Then,  one  may  inquire  how  many  times  it  mag- 
nifies. We  shall  now  proceed  to  show  by  a  simple  method  how 
one  may  satisfactorily  judge  of  the  defining  and  resolving 
powers  of  a  microscope. 

VII.     TESTING  THE  OPTICAL  POWERS. 

The  defining  and  resolving  powers  of  a  microscope  may  be 
best  tested  by  means  of  so-called  "proof  objects"  or  "test 
objects."  These  consist  of  small  parts  of  animals  or  plants, 
and  also  of  very  small  whole  organisms  which  are  prepared  in 
a  certain  way.  They  should  be  examined  with  a  known  magnifica- 
tion, which  should  be  produced  mainly  by  the  objective.  It 
should  then  be  ascertained  if  the  image  appears  the  same  in 
this  as  in  some  notoriously  good  microscope,  or  comparison 
should  be  made  with  some  distinct  and  clear  illustration  or 
exact  description  which  may  be  accessible.  If,  then,  one 
recognizes  all  those  details  which  the  illustration  or  description, 
with  a  like  magnification,  furnishes,  it  is  a  sign  of  the  good 
quality  of  the  microscope.  But  if  the  outlines  of  the  form  and 
the  fine  structural  relations  are  more  indistinct  than  in  the  illus- 
tration, or  if  they  are  in  general  not  visible,  it  would  thence 
follow  that  the  instrument  Would  not  fully  satisfy  modern  re- 
quirements. 

Since  the  testing  of  the  optical  performance  of  a  newly  ac- 
quired instrument  is  the  first  thing  to  be  taken  in  hand,  the 
maker  usually  takes  care  to  provide  some  test-object  by  which 
this  may  be  done. 

Testing  the  instrument,  particularly  with  objects  of  difficult 
resolution,  should  take  place  on  a  not  too  dark  day,  when  the 
heavens  are  uniformly  covered  with  a  veil  of  transparent  clouds  ; 


54  THE   MICROSCOPE   IN  BOTANY. 

at  all  events,  not  when 'the  sky  is  filled  with  numerous  gray,  rap- 
idly moving  clouds  which  produce  constant  changes  in  the  light. 
The  microscope  should  be  placed  close  before  an  open  window 
facing  the  north  or  east.  The  illumination  of  the  object  should 
be  by  central  light  from  the  mirror,  oblique  light  being  in  almost 
all  cases  unsteady.  The  size  of  the  aperture  in  the  diaphragm 
under  the  stage  to  be  employed  will  depend  upon  the  power 
of  the  objective  in  hand.  It  is  also  recommended  that  people 
who  use  spectacles  should  lay  them  aside  when  observing  test 
objects,  since  the  least  particle  of  greasy  matter,  such  as  might 
easily  be  left  on  the  glass  by  the  motion  of  the  eyelashes,  would 
render  the  microscopic  image  less  sharp  and  clear.  It  is  self- 
evident  that  the  objectives,  oculars  and  the  glasses  holding  the 
test-object  should  be  perfectly,  clean. 

We  will  first  discuss  those  objects  by  which  we  test  the  de- 
fining power  and  afterwards  those  by  which  we  test  the  resolv- 
ing power  of  the  microscope. 

A.    TESTING  THE  DEFINING  POWER. 

All  the  objects  used  for  this  purpose  in  the  magnifications 
employed  must  show  an  altogether  distinct,  clear,  delicate  and 
colorless  outline.  The  test-objects  used  exclusively  for  defini- 
tion should  be  employed  only  with  low  or  medium  powers, 
while  those  for  resolution  should  be  used  with  the  highest 
powers,  they  likewise  giving  at  the  same  time  tests  of  the 
definition.  The  most  important  tests  for  definition  are  the  fol- 
lowing : 

1.  The  Calcareous  Plates  of  the  Synapta.  That  part  of  the 
echinoderm  group  known  as  Holothurians  or  "sea-rolls"  con- 
tains animals  of  a  mostly  cylindrical  form  whose  bodies  are  cov- 
ered with  a  leathery  skin.  Imbedded  in  this  skin  are  a  great 
number  of  microscopically  minute,  perfectly  colorless  (rarely 
somewhat  colored)  calcareous  bodies.  The  genus  Synapta,  of 
which  representatives  are  found  in  the  Mediterranean,  but  whose 
more  numerous  species  occur  in  the  warm  seas  of  Polynesia  and 
southern  Asia,  shows  an  extraordinarily  delicate  example  of 
these  small,  calcareous  bodies.  The  integument  of  these  long 


TESTING  THE  DEFINING  POWER.  55 

worm-like  animals  is  pretty  thin,  and  growing  in  it  are  rectan- 
gular or  roundish,  perforated,  calcareous  plates  (Plate  I,  Fig. 
1,  b)  in  which  anchor-like  hooks  are  fastened  (Plate  I,  Fig.  1, 
a).30  The  fully  formed  calcareous  anchors  have  a  length  of 
0.97-0.98  mm.,  are  as  clear  as  glass  and  perfectly  transparent. 
The  end  of  the  handle  is  slightly  bifurcated  and  a  little  warty- 
rough.  The  anchor  hooks  are  curved  bow-like  and  bluntly 
rounded  at  the  end.  The  rectangular  little  plates  are  about 
0.8  mm.  long,  and  are  perforated  with  from  34  to  40  holes  great 
and  small,  the  larger  being  in  the  middle  and  the  smaller 
towards  the  ends.31 

The  anchors  of  the  Synapta  are  suitable  for  testing  the  re- 
solving power  of  very  low  objectives  only  (10  to  50  magnifica- 
tions). It  should  show  a  contour  in  this  magnification  sharply 
bounded  by  a  thick  black  line.  In  the  perforated  plates  this 
contour,  corresponding  to  its  greater  thinness,  is  more  delicate 
than  in  the  anchors.  Poor  glasses  show  an  imperfectly  defined 
outline  with  a  soft  haze  around  the  edges.  Lenses  imperfectly 
achromatic  produce  a  numercusly  colored  border  near  the  edges. 
The  illustration  in  Plate  I,  1,  is  prepared  from  a  magnification 
of  about  thirty  diameters.  The  specimen  should  be  mounted 
•  in  balsam. 

2.  Transverse  section  of  Coniferous  wood.  While  the  ob- 
jects already  described  are  suitable  only  for  testing  the  lower 
powers,  we  have,  in  a  transverse  section  of  the  needle-bearing 
trees,  a  very  good  object  for  testing  the  definition  of  medium,  and 
even  stronger  powers.  It  is  a  matter  of  indifference  which  one  of 
the  coniferous  species  one  chooses  from  which  to  make  the  prep- 
aration. The  one  illustrated  here,  Plate  I,  Fig.  2,  is  a  piece  of 
the  cross  section  of  the  young  stem  of  the  common  fir  tree 
(Pinus  sijlvestris)  which  may  be  found  anywhere.  The  prep- 
aration was  made  in  this  way.  The  most  delicate  possible 
section  was  made  through  the  young  stem.  Xo  dull  places  in 
the  knife  should  be  permitted  to  leave  their  marks  upon  the 

30  The  calcareous  bodies  from  the  following  and  other  species  of  the  Synaptae  are  suitable 
for  test-objects.    Synapta  inhaerens,  glabra,  Godefroyi,  recta,  similis,  molesta,  Kefersteinii, 
Besselii,  diyitata. 

31  There  are  also  species  (for  example,  S.  inhaerens')  in  which  the  calcareous  plates  have 
a  few  large  holes  with  toothed  edges. 


56  THE   MICROSCOPE   IN  BOTANY. 

section.  How  this  is  to  be  avoided  will  be  shown  more  exactly 
hereafter.  The  section  should  then  be  put  for  some  minutes  in 
absolute  alcohol  to  remove  the  resin  from  the  resin  tubes,3'2  then 
it  should  be  washed  in  distilled  water  and  mounted  in  glycerine- 
jolly  or  glycerine. 

For  our  purpose  we  shall  consider  the  wood  layer  which 
forms  one  concentric  ring.  With  high  magnification  the  wood 
cell33  presents  the  three  following  layers  in  its  walls.  The 
one  layer  which  is  common  to  the  two  cells,  and  which  we 
shall  with  Sachs  name  the  "middle  layer,"  a,  is  thin  and 
highly  refractive.  Then  follows  toward  the  inside  a  second 
stronger  layer,  which  is  compounded  from  several  concentric 
lamellae,  the  intermediate  thickening  layer  6,  and  upon  this  lies 
a  soft  layer  which  lines  the  inside  of  the  cell,  and  is  called  the 
inner  thickening  layer,  c.  All  these  layers  should  appear  clearly 
and  sharply  differentiated  from  each  other  with  a  linear  magnifi- 
cation of  from  450  to  800  diameters.  The  middle  layer  is  com- 
monly somewhat  easier  to  see  than  the  inner  thickening  layer. 
Hence  the  former  will  become  visible  earlier  when  the  prepara- 
tion is  put  under  the  lens,  and  the  latter  as  it  is  progressively 
subjected  to  higher  and  higher  powers.  The  inner  layer  is  best 
adapted  to  give  account  of  the  definition  of  high  power  lenses. 
With  good  objectives  the  inner  layer  should  show  itself  sepa- 
rated from  the  cell  cavity  in  sharp  outline,  a  very  sharp  delicate 
line  bounding  it.  Poor  objectives,  on  the  other  hand,  show  a 
broad  gray  line  about  the  cell  cavity  which  becomes  clearer  in- 
wards, with  no  sharp  limits  but  gradually  loses  itself  in  a 
delicate  haze.  It  should  be  noticed  that  thick  and  imperfect 
sections  produce  the  same  kind  of  an  image.  On  this  account, 
only  the  thinnest  possible  sections,  as  already  mentioned,  should 
be  used  for  this  investigation. 

o 

Longitudinal  sections  of  the  woody  parts  of  this  plant  furnish 
good  test-objects  for  these  magnifications  and  for  somewhat 
lower  powers.  Here  we  find  the  great  rounded  "  bordered 
pits"34  which  are  very  suitable  for  testing  the  definition  of  au 

32  Sach's  Lehrb.  clev  Bot.,  IV,  Aufl.  p.  95,  Fig.  78. 

33  Sachs,  1.  c.,  p.  75,  Fig.  57. 
s*  Sachs,  L  c.,  p.  25,  Fig.  23. 


TESTING  THE  RESOLVING  POWER.  57 

objective.     Their  entire  outline  should  appear  sharp  and  clear, 
as  simple  lines.35 

3.  Scale-dots  of  Lycaena.  We  may  for  the  same  purpose, 
with  good  results,  employ  the  scales  of  the  "Bluelings."  I  in- 
deed recommend  that  the  three  species  be  used  in  the  investi- 
gation :  Lycaena  Alexis  F.  (= Icarus  Hbst.),  L.  argiolus  L. 
or  L.  argus  L.  As  with  all  butterflies  their  wings  are  closely 
covered  with  numerous  small  stalked  scales,  to  which  they  are 
indebted  for  their  lively  colors.  On  the  upper  as  well  as  on  the 
under  side  of  the  wing  are  two  kinds  of  scales,  one  of  a  longer 
form  which  bears  on  its  surface  delicate  longitudinal  markings, 
and  another  which  consists  of  a  longer  style  joined  to  an  ellipti- 
cal plate.  The  latter  are  provided  on  the  upper  surface  with  a 
few  (6  to  10)  dotted  longitudinal  markings,  Plate  I,  Fig.  3. 
These  scales  sKould  be  mounted  in  Canada  balsam  for  test-objects. 
Good  medium  objectives  magnifying  35(Xto  450  diameters  should 
show  in  the  former  kind  the  longitudinal  markings  clearly  as 
double  lines.  And  in  the  latter  the  dots  should  appear  as  small 
circles  which  have  a  minute  dark  dot  in  the  middle.  The 
markings  and  the  dots  should  not  mingle  or  blend  with  each 
other.  The  specimen  is  somewhat  more  difficult  to  resolve 
when  mounted  dry,  than  in  Canada  balsam,  and  appears  colored. 


B.    TESTING  THE  RESOLVING  POWER. 

In  order  to  be  assured  of  the  superior  quality  of  the  higher 
and  highest  power  objective-systems  we  must  employ  the  best 
test-objects,  which  in  the  first  instance  permit  us  to  judge  of  the 
resolving  power,  and  at  the  same  time  are  a  test  of  the  definition. 
Both  qualities  of  the  objective  are  essentially  bound  up  to- 
gether. An  objective-system  with  unsatisfactory  definition  will 
never  resolve  difficult  images.  But  it  sometimes  happens  that 
two  systems  of  very  nearly  the  same  resolving  power,  showing 
the  resolved  details  in  sharper  or  fainter  outlines,  do  not  possess 

83  Preparations  of  potato  starch  grains  maybe  employed  as  tests  of  definitions  for 
medium  powers.  They  should  be  examined  in  water  or  glycerine,  and  their  separate 
layers,  which  are  grouped  about  an  excentric  formation-point,  should  be  bounded  by  a 
sharp,  strong  and  delicate  outline.  (See  Sachs,  1.  c.  Fig.  51.  on  p.  62.) 


58  THE   MICROSCOPE   IN  BOTANY. 

• 
the  same  power  of  definition.     The  test-objects  most  often  used 

for  resolution  are  butterflies'  scales  and  the  siliceous  frustules 
of  different  species  of  diatoms. 

1.  Scales  of  Hipparchia  janira  and  Lycaena  argiolus.  Our 
common  white  butterfly,  Hipparchia  janira,  has  on  its  wings 
several  kinds  of  scales,  short,  medium  and  long.  A  scale  of 
medium  length  is  illustrated  in  Plate  I,  Fig.  4.  It  has  a 
breadth  of  0.059  mm.,  and  a  length  of  0.156  mm.  It  is  rec- 
tangular, has  three  broad  points  at  top,  and  is  heart-shaped  at 
bottom,  ending  in  a  short  style.  Its  surfaces  are  covered  with 
22-24  longitudinal  ridges,  which  have  an  average  distance  apart 
of  0.00266  mm.,  so  that  about  four  of  these  go  to  0.01  mm. 
(10/Jt  =10  micromillimetres).  Magnified  as  in  the  illustration 
(305  diameters)  we  see  very  many  delicate  cross  lines  between 
the  longitudinal  elevations.  The  higher  the  magnification,  the 
more  the  finer  details  are  brought  out,  and  the  butterfly's  scale 
therefore  furnishes  a  very  excellent  preparation  for  testing  the 
resolving  power  of  the  strongest  objective-system.  In  recent 
times,  Dipper36  has  most  accurately  investigated  the  butterflies' 
scales  employed  as  test-objects,  and  we  shall  give  here  in  his 
own  words  the  results  to  which  that  naturalist  has  arrived 
in  his  very  exact  studies  of  these  important  tests.  "The 
longitudinal  flutings  are  made  by  the  elevation  of  the  upper 
surface,  between  which  run  furrow-like  depressions  so  that  the 
scale  seen  in  section  has  a  wavy  aspect.  When  viewed  with 
lower  powers,  and  oblique  light  falling  upon  them  in  a  direction 
perpendicular  to  their  length,  they  appear  to  be  bounded  by 
two  sharp  lines.  With  higher  magnification  and  central  light, 
and  with  an  objective  of  good  definition,  they  assume  a 
toothed  appearance,  and  because  of  the  cross-lines  which  lie 
in  the  same  plane,  and  come  into  the  focus  at  the  same  time, 
they  take  the  appearance  of  being  thickened  at  these  points. 
With  the  lower  powers,  this  structural  relationship,  on  ac- 
count of  its  delicacy,  shows  with  scarcely  half  the  sharpness 
of  the  boundary  lines.  With  oblique  illumination,  even 
with  the  medium  higher  powers,  it  is-  overlooked,  because  the 
shadows  cast  by  the  longitudinal  ridges  apparently  obscure 

36  Dippel,  /.  c.,  p.  118 /. 


TESTIXG  THE  RESOLVING  POWER.  59 

these  fine  lines.  Herein  we  see  the  foundation  for  the  widely 
divergent  views  which  microscopists  have  expressed  about  the 
real  nature  of  these  longitudinal  markings.  Thus,  for  example, 
Brewster  (Treatise  on  the  Microscope)  declared  that  the  cross 
markings  did  not  exist  at  all,  but  that  the  longitudinal  ridges 

c5  O  O 

were  beset  with  small  teeth.  Chevalier  (Les  Microscopes, 
etc.,)  described  the  scales  of  Pieris  brassicae  as  beset  with 
longitudinal  ridges  which  were  formed  by  minute  balls  placed 
near  each  other,  and  held  that  the  true  test  of  an  objective- 
system  consisted  in  making  these  globules  visible.  Some 
English  microgra  pliers  agreed  with  this.  Others,  for  example, 
Goring,  then  under  the  German  H.  v.  Mohl,  controverted  it, 
and  asserted  the  existence  of  sharply  defined  longitudinal  and 
transverse  markings,  and  held  Chevalier's  description  of  this 
test-object  to  be  a  plain  witness  that  the  latter  had  misinter- 
preted his  microscope.  Brewster  is  only  partly  in  the  right 
with  his  statement,  since  he  quite  overlooked  the  cross-lines, 
or  rather  failed  to  see  them  altogether ;  but  Chevalier  decided 
correctly.  I  have  examined  the  same  object  anew,  with 
several  of  the  best  objective-systems  of  recent  times  and 
find  them  formed  as  Chevalier  asserted.  ,  The  well-known  heart- 
shaped  scales  of  Pieris  brassicae37  are,  especially  over  their 
upper  surfaces,  both  on  the  longitudinal  markings  and  interven- 
ing spaces,  beset  with  small,  irregularly  angled,  or  roundish 
bodies,  whereby,  under  certain  conditions  of  illumination  the 
appearance  of  cross-lines  is  produce.d,  which  run  between  and 
near  the  longitudinal  lines,  but  which  by  direct  illumination 
and  good  defining  objectives  are  seen  to  appear  in  the  way 
pointed  out  by  Chevalier. 

In  most  scales,  the  cross  markings  run  in  a  direction  perpen- 
dicular to  the  axis  of  the  longitudinal  ridges ;  in  others,  in  an 
oblique  direction,  as  well  over  the  summits  of  them,  as  across 
the  intervening  spaces,  without  interruption.  In  those  adjust- 
ments of  the  microscope,  however,  with  which  one  commonly 
chooses  to  see  the  cross  marking  between  the  others  with  dis- 
tinctness, these  facts  easily  escape  observation.  Only  by  a 
certain  medium  adjustment  do  they  come  forth  clearly.  The 

37  See  Dippel,  1.  c.,  Figs.  61  and  62,  on  p.  119. 


60  THE   MICROSCOPE   IN   BOTANY. 

cross  markings  are  not  like  the  longitudinal,  elevations,  but 
rather  depressions  between  the  more  or  less  regularly  rectangu- 
lar to  roundish  bodies,  which  as  a  rule  stand  in  series  of  four 
between  each  two  of  the  longitudinal  markings.  Thence  be- 
tween each  two  of  the  stronger  longitudinal  markings,  there  are 
three  others  very  much  more  delicate,  and  which  are  far  more 
difficult  to  see  than  the  transverse  markings,  and  afford  good 
tests  of  the  strongest  lenses.  The  best  objective-system  in 
which  is  united  the  greatest  resolving  power  with  the  best 
definition  as  well  as  the  most  perfect  chromatic  correction,  can 
alone  make  us  acquainted  with  this  structure.38  Bruno  Hasert 
had  already,  in  1847,  traced  out  this  structure  and  since  then 
more  fully  examined  it.  (Official  report  of  the  34th  meeting  of 
German  Naturalists  and  Physicians,  at  Karlsruhe,  p.  212.)  At 
his  suggestion,  I  have  likewise  most  carefully  examined  this 
object  with  my  most  powerful  objectives,  and  have  convinced 
myself  definitely  of  the  correctness  of  his  representation.  Tims 
we  have  established  a  solid  footing  as  to  how  these  cross 
markings  ought  to  look  through  good  objectives.  In  the  same 
way  we  explain  the  diagonal  markings  on  these  scales,  which 
one  perceives  with  certain  illuminations  and  with  strong  objec- 
tives. If  we  examine  the  transverse  lines  with  direct  light  and 
a  magnification  of  300  to  500  diameters  they  will  appear  as  if 
serrated,  but  they  must  be  sharply  defined  for  that.  With 
higher  magnifications  the  separate  little  bodies  will  appear  with 
clear  delicate  outlines,  as  soon  as  the  spherical  aberration  of 
the  objective  is  perfectly  corrected.  Oblique  illumination,  on 
the  contrary,  is  the  cause  of  obscuring  the  true  structure  with 
low  magnifications,  and  affords  sharply  marked  linear  cross 
stripes,  as  they  have  been  heretofore  represented  by  those  mi- 
croscopists  who  have  in  such  testing  worked  with  their  mirrors 
excentrically  placed.  Objectives  also  which  are  not  strictly 
first-class  with  respect  to  definition,  or  have  imperfect  correc- 
tion of  chromatic  aberration  will  give  that  kind  of  an  imnge, 
with  high  magnifications.  On  this  we  may  ground  objections  to 
a  system  which  is  known  to  be  otherwise  excellent,  that  it  shows 
the  cross  markings  of  the  Hipparchia  scale  as  serrated.  On  the 

38  See  Plate  I,  Fig.  7,  which  represents  a  copy  of  Dippers,  Fig.  G9. 


TESTING  THE  RESOLVING  POWER.  61 

contrary,  we  must  accept  it  as  evidence  of  the  good  quality  of 
an  objective,  that  it  shows  these  markings  with  central  illumi- 
nation quite  distinct  and  sharp,  while  one  which  does  not  thus 
show  them  betrays  a  lack  of  defining  power.  The  diagonal 
lines  come  out  when  the  oblique  light  falls  upon  the  scale  at  an 
angle  of  30°  to  60°  to  the  axis  of  the  longitudinal  markings, 
while  the  delicate  longitudinal  lines  come  out  most  clearly 
when  the  oblique  light  falls  perpendicularly  upon  the  longitudi- 
nal axis.  In  respect  to  the  visibility  of  the  two  last  named 
systems  of  lines,  the  butterflies'  scales  furnish  test-objects  of  a 
difficulty  almost  equal  to  the  diatoms,  without,  however,  afford- 
ing a  sufficiently  perfect  series  of  comparisons." 

It  should  be  added  that  Plate  I,  Figs.  5,  6,  and  7,  shows  us 
single  pieces  of  the  scale  of  Hipparchia  janira  under  different 
magnifications,  Fig.  5  X  500,  Fig.  6  X  1450,  Fig.  7  X  1920 
times,  the  last  two  after  Dippel.39 

Lycaena  argiolus  has  scales  which  are  somewhat  more  difficult 
to  resolve  than  those  of  Hipparchia  janira  in  respect  to  their 
corresponding  fine  markings.  Fig.  8,  Plate  I,  represents  a 
whole  scale  x  305  times.  Fig.  9,  a  piece  X  500,  and  Fig.  10, 
a  small  piece  X  1450  times  (after  Dippel).40 

Butterfly  scales  are  commonly  mounted  in  Canada  balsam  for 
test-objects.  Mounted  dry  they  are  somewhat  more  difficult. 
Glycerine  mounting  seems  to  me  very  serviceable  also. 

2.  The  Siliceous  frastides  of  the  diatoms.  There  occurs  in 
the  mire  and  on  plants  in  stagnant  waters,  and  also  in  the  sea, 
a  group  of  single-celled  algse  in  an  almost  endless  variety  of 
forms  and  species,  known  in  general  as  diatoms.  They  are 
throughout  of  microscopical  minuteness,  and  are  distinguished 
from  all  other  algae  by  having  their  cell  walls  covered  with  a 
framework  of  pure  silex,  which  consist  of  frustules  that  fit 
upon  each  other  and  represent  a  perforated  lattice- work  of  the 
most  delicate  structure.  This  siliceous  shield  consists  of  two 
separable  halves.  If  the  diatoms  are  boiled  in  a  mixture  of 
potassium  chlorate  and  nitric  acid  the  entire  organic  substance 
will  be  destroyed  and  only  the  siliceous  frames  will  be  left  over 

so  Dippel,  1.  c.,  Figs.  63  and  69,  on  p.  119  and  122. 
*>Dii  pel,  1.  c.,  Fig.  73,  0.1  p.  1-23. 


62  THE  MICROSCOPE  IN   BOTANY. 

with  the  two  halves  parted.  These  separated  halves  of  the 
frustules  are  what  furnish  the  most  excellent  test-objects. 
They  are  prepared  in  two  ways,  either  mounted  dry  or  in  bal- 
sam, and  the  method  of  examination  as  test-objects  is  determined 
by  the  particular  kind  of  mounting.  On  the  whole,  those 
mounted  in  balsam  are  more  difficult  to  resolve  than  those 
mounted  dry. 

Dry  mounts  are  most  suitably  made  as  follows.  After  the 
diatoms  have  been  separated  by  the  mixture  of  nitric  acid  and 
concentrated  solution  of  potassium  chlorate,  the  fluid  containing 
the  diatoms  is  put  into  a  high  and  narrow  test  tube,  and  the 
diatoms  are  allowed  to  settle  to  the  bottom.  They  are  then 
repeatedly  washed  in  distilled  water  till  litmus  paper  no  longer 
shows  any  trace  of  the  acid.  A  sample  of  the  diatomiferous 
fluid  is  taken  out  with  a  pipette,  and  placed  on  a  clean  slide,  and 
the  water  allowed  to  evaporate  in  some  place  free  from  dust, 
(for  example  in  a  drying  chamber)  and  a  cover  glass  cemented 
on  over  it,  as  will  be  described  further  along.  The  balsam 
mount  is  made  in  the  following  way.  A  small  portion  of  the 
cleaned  diatomiferous  fluid  is  put  in  a  watch  glass  and  evapo- 
rated. The  residue  is  mixed  with  pure  oil  of  turpentine,  and 
then  a  drop  of  the  turpentine  oil  with  the  suspended  diatoms  is 
mingled  with  a  drop  of  Canada  balsam  on  a  slide  ;  lay  on  a  cover 
glass  and  fasten  with  gentle  warming.41 

Several  species  of  the  genus  Pinularia  make  tests  for  very 
low  powers.  The  cross  markings  on  the  long  sausage-shaped 
frustules  of  Pinularia  nobilis  Eh.  can  be  easily  and  clearly  seen 
with  a  magnifying  power  of  thirty  diameters,  but  the  like  form- 
ations on  the  elongated  elliptical* body  of  P.  viridis^Rh.  must 
be  magnified  209  diameters  to  be  distinctly  seen.  Since  we 
have  shown  how  the  butterfly  scales  may  most  suitably  test  the 

41  Except  in  rare  cases,  we  shall  not  ourselves  undertake  the  preparation  of  the  diatom 
test-objects.  They  can  be  had  at  the  best  microscopical  institutes  (Dr.  Kaiser  in  Berlin, 
Mollerin  Wedel,  Holstein,  Rohdig  in  Hamburg,  etc.)  [In  America  all  of  the  principal 
dealers  in  microscopical  goods  keep  diatom  tests  on  sale,  and  I  believe  Mr.  C.  L.  Petico- 
las  of  Richmond,  Va.,  has  made  a  specialty  of  the  preparation  of  these  diatom  test-objects, 
A.  B.  H.|  at  the  price  of  one  to  two  Marks  each  (25  to  50  cts.)  Moller  furnishes  a  diatom 
test  plate  which  contains  under  the  same  cover-glass,  a  number  of  diatom  frustules  for 
test-objects  so  arranged  that  tiiose  at  one  end  are  easiest,  and  they  become  progressively 
harder  of  resolution  towards  the  other  end,  so  that  it  is  possible  to  test  the  power  of  resolu- 
tion of  all  systems  by  means  of  this  one  preparation. 


TESTING  THE  RESOLVING  POWER.  63 

lower  powers,  we  need  not  further  consider  the  diatoms  as  tests 
for  objectives  of  that  kind. 

For  objective-systems  of  medium  magnifications,  the  various 
species  of  the  genus  Pleurosigma  make  most  extraordinarily 
beautiful  test-objects.  They  are  almost  all  good  tests  also  for  the 
higher  powers.  The  species  of  the  Pleurosigma  are  easily  recog- 
nized by  the  peculiar  sigma-like  (?)  curvature  of  their  bodies. 
A  doubly  curved  central  line  is  drawn  along  their  whole  length 
which  in  the  middle  is  expanded  into  a  lengthened  nodule.  On 
each  side  of  the  central  line,  the  frustule  is  covered  throughout 
with  a  framework  of  delicate  lines,  which  now  lay  claim  to  our 
exclusive  attention.  The  species  used  as  test-objects,  may  be 
separated  into  two  groups  according  to  the  construction  of  the 
above  mentioned  framework  or  skeleton.  The  first  includes 
the  species  Pleurosigma  balticum  and  PL  attenualum;  to  the 
second  belong  PL  angulatum  and  PL  formosum. 

(a)  We  shall  first  consider  Pleurosigma  balticum  Sm.  This 
little  plant  is  from  0.29  to  0.33  mm.  long,  and  should  be 
mounted  in  Canada  balsam  when  used  as  a  test  object.  Plate 
I,  Fig.  11-14.  By  a  magnification  of  100  diameters  the  object 
appears  as  a  delicate  hyaline  form,  of  the  shape  of  Fig.  11.  We 
distinguish  the  lateral  boundary  edges,  and  the  beautifully 
curved  central  line  with  a  knot  in  the  middle.  With  this  mag- 
nification we  may  perceive  either  by  central  or  oblique  light, 
minute  carvings  on  the  surface  of  the  frustules.  If  the  magnifi- 
cation be  now  raised  to  200  diameters,  by  means  of  a  stronger 
objective  and  the  weakest  possible  ocular,  the  surface  will 
appear  to  be  covered  with  a  very  delicate  lattice-work.  We 
see  at  once  that  this  consists  of  longitudinal  and  transverse 
striae.  By  different  focussing,  now  the  former,  and  now  the  lat- 
ter, will  be  made  distinctly  visible,  particularly  towards  the 
outer  edges.  Now,  if  we  choose  a  higher  ocular,  taking  the 
same  objective,  and  make  the  magnification  300  diameters,  the 
image  will  become  a  little  clearer  since  the  single  lines  will  be 
farther  apart.  Two  transverse  lines  stand  at  a  distance  of 
0.0007  mm.,  so  that  about  fifteen  of  them  go  to  make  0.01  mm. 
An  objective  magnification  of  about  460  clearly  resolves,  the 
lattice-work  into  two  systems  of  lines  which  stand  perpendicu- 


64  THE   MICROSCOPE   IN  BOTANY. 

lar  to  each  other,  one  running  lengthwise  and  the  other  across 
the  diatom.  We  now  without  difficulty  see  that  at  the  inter- 
secting points  of  the  atrice  of  the  two  systems  are  knot-like 
thickenings,  Plate  I,  Figs.  12,  13,  the  form  of  which  is  appar- 
ently rectangular. 

A  little  stronger  ocular  magnification  (550  to  590)  Fig.  12, 
13,  makes  the  image  still  somewhat  more  distinct.  Finally, 
the  markings  appear  to  be  perfectly  resolved  with  a  magnifica- 
tion of  950,  Plate  I,  Fig.  14.  The  knots  have  now  clearly  a  four- 
sided  form,  and  by  a  still  stronger  magnification  (1400  to  1450) 
we  recognize  the  fact  that  they  are  in  reality  six-sided,  but  the 
six  angles  are  not  regular,  two  opposite  sides  being  shorter 
than  the  other  four.42 

(b)  Pleurosigma  angulatum  Sm.  forms  the  diatom  test 
which  is  properly  most  in  use  for  medium  and  higher  powers. 
They  should  be  mounted  dry.  If  mounted  in  Canada  balsam, 
they  are  considerably  more  difficult  of  resolution,  and  the  fol- 
lowing description  would  not  apply  to  a  balsam  mount.  PL  an- 
gulatum, which  may  easily  be  distinguished  from  all  other  species 
of  the  genus  by  its  form,  attains  a  mean  length  of  0.24  to  0.32 
mm.  Both  sides  of  the  middle  portion  of  the  frustule  are  drawn 
out  somewhat  sharply  angular,  Plate  II,  Fig.  1,  whence  it  gets 
its  name.  The  middle  rib  differs  but  little  from  PL  balticum 
only  being  a  little  slenderer  and  lacking  the  slight  curvature 
each  side  of  the  central  nodule.  With  low  magnification  PL 
angulatum  has  a  bright  yellow-brown  color.  We  shall  now 
examine  the  structure  of  the  frustule  of  this  diatom  with  low, 
medium,  higher  and  highest  magnification. 

1.  Low  powers  (50-150  diameters).  The  surface  shows 
a  perfectly  homogeneous  aspect.  The  color  is  uniformly  clear 

42  To  those  who  are  disposed  to  undertake  the  testing  of  microscopes  for  themselves  by 
means  ol'these  objects,  we  especially  commend  the  work  of  Fritschnnd  MUller.  -'The  carv- 
ings and  finer  structural  relations  of  the  Diatomacece,  with  reference  to  the  use  of  the  spec- 
ies as  test-objects.  Part  I,  12  plates,  photo-micrographic  illustrations,  Berlin,  1370,  4to. 
Price  16  Marks,  each  plate  singly  1.6  Marks."  The  plates  represent  beautiful  photographs 
direct  from  the  microscope :  I.  Diatom  type  plate,  No.  II  of  J.  D.  Moller  in  Wedel,  magnified 
90  diameters.— -II.  Arachnodiscus  ornatus  Ehrbg.  X  530.—  III.  Triceratium  favus  Ear.  X 
545.-  IV.  Pinularia  nobilis  Kg.  X  545.—  V.  Navicula  Lyra  Ehr.  and  var.  X  530.—  VI. 
Stauroneis  P/teemce??*mw  Ehr.X545.—  VII.  Pleurosigma  balticum  Sm.X545.—  VIII  and  IX. 
PI.  angulatum  Sm.  X  515,  1200.—  X.  Grammatophora  marina  Sm  X  545.— Gram,  oceanica 
Ehr.  =  G.  subtitissima;  X  "00.—  XI.,  XII.  Surarella  gemma  Ehr. ;  X  ^co,  1200. 


TESTING  THE  RESOLVING  POWER.  65 

brown.  Oblique  illumination  reveals  no  further  details  on  the 
surface.  The  diatom  cannot  be  used  as  11  test  for  lower  powers. 

2.  Medium  powers     (200-400    diameters).     With  a  mag- 
nification of  200  to  250  diameters,  produced  in  conjunction  with 
the  weakest   ocular,   we   first   recognize   traces    of  markings, 
Plate  II,  Fig.  1,  which  come  forth  in  a  dark  brownish  shade. 
Concerning  the  nature  of  these  markings,   this   magnification 
permits  us  to  say  nothing  further .     But  if  one  raises  the  mag- 
nification by  a  somewhat  stronger  ocular,  to  about  300  diarn*- 
eters,    and   uses    oblique   illumination,  the  markings  will   be 
distinct,  and  recognized  as  consisting  of  three  systems  of  stria-,. 
which  are  inclined  to  each  other   at  an  angle  of  about  120°. 
With  this  magnification,    however,  with  the  use  of  oblique  ilr 
lumination,  we  recognize  these   systems   only   when  the  light 
falls  upon  them  severally  at  an  angle  of  90°.     Concerning  this 
quality  which  it  is  well  to  regard,  Dippel43  first  remarked :  "If 
the  oblique  light  be  applied  to  one  side,  there  will  appear  one 
or  the  other  of  the  systems  of  lines  according  to  the  situation  of 
the  diatom.     When  the  oblique  light  falls  parallel  with  its  long- 
est axis  the    somewhat  more  widely  separated  cross-lines  will 
appear.     If  now  we  turn  the  light  about  90°,  the  more  closely 
drawn  diagonal  systems  of  lines  will  appear,  with  somewhat  the 
same  sharpness.     On  the  contrary,  only  one  of  these  systems  of 
lines  will  become  sharply  visible   when  the  longitudinal  axis  of 
the  frustule  forms  an  an^le  of  about  45°  with  the  direction  of 

C 

the  rays."  Condenser  illumination,  without  stopping  off  the 
central  rays,  gives  on  the  whole  a  much  clearer  picture  than 
the  above  described  process. 

3.  Higher  magnifications  (450  to  900  diameters).     When 
the  objective  magnification  reaches  450  to  480  diameters,  the 
three  systems  of  lines  on  the  surface  of  the  frustule  become 
distinctly  visible,  mutually  crossing  each  other  at  an  angle  of 
120°,  Plate  II,  Fig.  2.     But  we  next  observe  that  what  at  first 
appeared  to  be  striae  are  not  straight  lines,  but  that  they  are 
hexagons  lying  side  by  side  in  rows  in  the  same  direction,  which 
cover  the  whole  surface  of  the  diatom  with  the  most  extraordi- 
narily delicate  lattice-work,  Plate  II,  Fig.  4.     With  a  magiiiti- 

«  Dippel,  Das  Mikroskop.  Bd.  I,  p.  12S. 
5 


66  THE  MICROSCOPE  IN  BOTANY. 

cation  of  450  to  480  diameters,  the  hexagons  can  be  seen  distinctly 
only  when  the  light  is  central  and  the  focussing  upon  a  given 
place  is  extremely  sharp.  If  the  illumination  is  excentric  some 
one  system  will  prevail,  according  to  the  direction  in  which  the 
lattice-work  is  illuminated,  as  is  indicated  above.44  Especially, 
with  central  condenser  illumination,  the  image  becomes  very 
clear.  By  right  focussing,  the  little  hexagonal  surfaces  will 
appear  quite  colorless,  and  their  sharply  bounded  contour  some- 
what chocolate  colored.45 

When  the  image  is  produced  with  a  good  objective,  it  ought 
to  bear  considerable  ocular  magnification  without  impairing  its 
distinctness.  Produced  with  a  good  Gundlach's  immersion- 
system,  VIII,  the  image  bore  an  ocular  magnification  of  1000  to 
1400  diameters,  still  showing  the  corners  of  the  hexagon  per- 
.fectly  angled  and  sharply  defined. 

4.  The  highest  magnifications  (900-2000  diameters). 
With  magnifications  of  over  900  the  resolution  of  the  hexagonal 
.network  will  become  still  more  distinct.  Since  the  true  diam- 
eter of  the  hexagon  is  about  0.005  mm.,  it  is  evident  that  it  may 
be  very  distinctly  perceived  when  magnified,  we  will  say,  to  1000 
times.  We  have  repeatedly  studied  PL  angulatum  with  the 
Seibert immersion-system,  No.  IX  (x  950,  1430,  2170,  2880), 
and  have  seen  its  minutest  structure  with  central  and  oblique 
illumination  and  with  the  use  of  all  oculars ;  and  most  beauti- 
fully by  the  use  of  a  condenser  with  the  middle  rays  shut  out. 
Plate  II,  Fig.  4,  shows  the  image  with  a  magnification  of 
2880  with  the  above  mentioned  system  ;  Fig.  3,  with  a  linear 
enlargement  of  1200,  the  latter  from  the  photo-micrograph  of 
Fritsch,  Plate  IX. 

The  species  of  the  genera  Grammatophora  and  Nitzschia  are 
connected  with  those  of  Pleurosigma  in  affording  the  very  best 
test  objects  for  the  higher  and  highest  powers  of  the  microscope, 
as  is  also  Surirella  gemma  whose  delicate  siliceous  frustule  can 
be  resolved  only  by  the  highest  and  best  magnification. 

4*  This  unequal  prevalence  shows  very  well  in  Plate  VIII  of  the  above  cited  work  of 
Fritsch  and  Muller  which  represents  a  Pleurosigma  angulatum  with  excentric  illumination. 
The  photograph  should  be  examined  in  different  places  with  a  good  magnifying  glass. 

45  By  wrong  focussing,  exactly  the  contrary  effect  is  produced ;  the  surfaces  are  dark  and 
the  contour  bright. 


TESTING  THE  RESOLVING  POWER.  67 

(c)  Grammatophora  marina  Sm.  The  body  of  the  Grammat- 
ophora  species  can  be  compared,  as  to  its  form,  with  nothing 
better  than  a  cigar  case.  The  exterior  aspect  of  this  genus  of 
diatoms  presents  the  form  of  a  more  or  less  elongated  rectangle 
with  the  angles  blunt  or  rounded,  Plate  II,  Figs.  5,  7.  Next 
to  the  middle,  run  two  zigzag-like  bent,  coarse  lines,  whose 
form  and  position  vary  according  to  the  species.  Outside  of 
these  longitudinal  lines,  on  the  whole  extent  of  the  outer  border, 
runs  a  zone  which  is  filled  with  the  most  delicate  transverse  striae. 
These  exclusively  claim  our  attention  here. 

If  we  examine  the  Gr.  marina  (in  Canada  balsam)  with  a 
magnification  of  200  diameters,  the  cross  lines  spoken  of  are 
scarcely  visible.  We  shall  see  them  only  after  looking  a  long 
time.  The  markings  beceme  somewhat  more  distinct,  with  the 
use  of  excentric  illumination,  or  better  still  with  condenser  il- 
lumination. The  weaker  ocular  magnifications  change  the  im- 
age but  very  little,  but  if  it  be  raised  to  about  600  diameters, 
and  excentric  illumination  be  used,  every  cross  line  will  appear 
to  consist  of  a  clear  bright  and  a  dark  or  shadowed  line. 

If  now  wre  raise  the  objective  magnification  to  450—480  diam- 
eters, the  markings  become  very  distinct,  both  by  central  and 
central-condenser  illumination.  Light  which  falls  upon  them 
obliquely,  and  especially  in  a  direction  perpendicular  to  the 
markings,  brings  out  the  image  much  more  distinctly.  With 
central  illumination  and  very  high  focussing,  the  striated  part 
of  the  diatom  appears  to  be  covered  with  minute  points  ;  deeper, 
the  cross  lines  seem  to  be  granulated.  The  image  is  most  distinct 
when  the  focus  is  made  to  touch  the  middle  between  the  upper 
and  lower  surface  of  the  diatom.46  Fig.  6  represents  Gr.  marina 
magnified  600  diameters. 

Stronger  magnifications  (900  to  1200)  change  the  image  but 

46  After  a  very  exact  study  of  the  Grammatophora  species,  I  here  express  the  view  that 
the  transverse  strife  are  formed  of  a  series  of  granular  elevations  standing  one  behind  an- 
other. In  this  1  come  into  controversy  with  Dip|»el  who  supposes  that  besides  the  smooth 
transverse  striation,  there  are  other  systems  of  lines  which  lie  over  these.  "In  all  other 
species,  there  occurs,  as  in  PI.  angulatum,  along  with  the  transverse  strice,  another  diagonal 
system  of  lines  which  cross  these,  and  which  in  Gr.  subtilissima  are  extremely  difficult  to 
see,  and  for  this  resolution  it  is  necessary  to  have  the  most  favorable  illumination  by  the 
use  of  well  regulated  oblique  light."  (Dippel.  /.  c..  Bd.  I,  p.  T29.)  I  hold  on  these  grounds 
that  Dippel's  illustrations,  Figs.  87-90,  are  not  altogether  true  to  nature.  Compare  them 
with  Fritsch  and  Moller,  I.  c..  Plate  X. 


68  THE  MICROSCOPE  IN  BOTANY. 

little.     Gr.  marina  is  principally  recommended  as  an  object 
for  the  weaker  immersion  systems. 

(d)  Grammalophora    oceanica  Ehrbg.:=6rr.    subtilissima . 
This  species  is  distinguished  from  the  one  described  above  by  its 
differing  form  (it  is  slenderer  and  longer  than  Gr.  marina)  and 
by  the  considerably  greater  fineness  and  difficulty  of  resolution 
of  the  transverse  fitrice.     While  the  transverse  lines  of  Gr.  ma- 
rina stand  about  0.00041  mm.  apart,  those  of  Gr.  oceanica  are 
removed  but  about  0.0003 1  mm.  from  each  other.     An  objective 
magnification  (dry  lens)  of  200  diameters  does  not  resolve  the 
markings  in  the  least,  neither  by  the  use  of  central,  oblique, 
or  condenser  illumination,  nor   even   if  the   magnification  be 
raised  by  means  of  oculars  to  400  or  500  diameters.     The  same 
thing  happens  with  the  lowest  immersion  system  whose  lowest 
ocular  magnification  does  not  exceed  400   or  500.     It  is  only 
when  the  magnification  reaches  700  with  a  low  ocular  that  the 
cross  bars  appear  in  the  form  of  very  delicate  lines,  Plate  II, 
Fig.  7.     With  still  higher  immersion  systems,  1200  to  1500  di- 
ameters, they  appear  still  more  distinct  and  differ  in  no  import- 
ant way   from   those  of  the  species  described  above.      Fig.  8 
shows  this  in  a  balsam  preparation. 

(e)  We  may  briefly  make  mention  here  of  a  very  good  test- 
object  from  another  genus  of  diatoms,  Nitzschia  linearis,  Plate 
II,  Figs.  9,  10.   It  has  a  peculiar  wand-like  form,  deeply  chan- 
nelled on  the  outer  edge.     Proceeding  from  this  there  are  drawn 
over  the  whole  surface  transverse  lines,  which  in  respect  to  del- 
icacy, and  distance  from  each   other,    stand    between  the  two 
species  of  Grammatophora  (0.00036  mm.).     A  clear  resolution 
of  these  can  be  reached  only  by  the  strong  immersion-systems. 
Fig.  10,  Plate  II,  represents  likewise  a  balsam  preparation. 

(f)  An   altogether  superior  diatom  test-object,  which  can 
be  perfectly  resolved  only  by  means  of  the  most  powerful  sys- 
tems, is  the  Surirella  gemma  Ehrbg.     It  should  be  mounted  dry, 
and  even  in  that  condition  it  is  extraordinarily  difficult  to  resolve. 
Plate  II,  Figs.  11-13,  represents  a  view  of  it  magnified  by  object- 
ive-systems 500,  700,  and  1200  times.     The  form  of  the  j$u,ri- 
rella  is  oval  and  the  ends  are  but  a  little  pointed.     The  oval 
surface  is  marked  over  with  a  framework  of  strong  siliceous 


TESTING  THE  RESOLVING  POWER.  69 

bars  which  run  across,  at  one  side  are  quite  irregularly  joined 
to  the  thickened  border,  and  on  the  other  to  the  longitudinal 
middle  line.  In  the  fields  between  these  bars  not  the  least 
trace  of  markings  is  discernible,  even  with  a  magnification  of 
500  diameters.  See  Fig.  11.  With  an  objective  magnification  of 
nearly  700,  lines  become  visible  in  the  fields  which  run  parallel 
with  the  cross  bars.  See  Fig.  12.  Indistinctly  with  this  mag- 
nification, but  clearly  with  one  of  1200  to  1500  diameters,  these 
lines  appear  to  consist  of  small  dots.  The  whole  field  makes 
the  impression  as  if  it  were  filled  with  a  basket-like  tissue.  See 
Fig.  13.  This,  according  to  Dippel,47  gives  ground  for  the  suppo- 
sition that  over  the  continuous  cross  lines  which  are  relatively 
strong,  run  very  delicately  drawn  longitudinal  lines,  the  latter 
being  seen  only,  for  the  most  part,  with  oblique  illumination. 
"These  diatoms  give  a  right  beautiful  image  when  the  latter 
method  of  illumination  is  used,  and  when  the  rays  touch  the- 
longitudinal  strice  at  about  an  angle  of  25°  to  30°." 

We  herewith  conclude  the  series  of  objects  which  serve  us  as 
the  best  tests  for  the  optical  powers  of  the  microscope.  The  most 
important  of  these  are,  however,  the  scales  of  the  Hipparchia 
Janira  and  the  frustules  of  Pleurosigma  angulatum.  To  these 
therefore  we  have  devoted  the  most  particular  description. 
Still  we  might  here  add  a  remark  as  to  test-objects  in  general. 
Many  dealers  furnish  test  objects  with  cover-glasses  0.15  to 
0.20  mm.  thick.  We  esteem  these  of  no  practical  use.  For  if 
we  undertake  to  use  one  of  them  with  very  high  powers,  we 
shall  find  the  cover-glass  so  thick  as  to  prevent  our  focussing 
down  to  the  object  itself,  and  so  in  spite  of  the  correction  screw 
the  image  would  be  ruined.  We  should  follow  the  lead  of 
Moller,  and  mount  test  objects  under  a  cover-glass  not  more 
than  0.05  to  0.08  mm.  thick.48 

To  Leopold  Dippel  belongs  the  credit  of  having  exactly  de- 
termined the  direct  distance  between  these  transverse  or 
longitudinal  markings  upon  the  scales  of  the  butterflies'  wings, 
the  diatoms,  etc. 

In   furtherance  of  our  present  aim  we  give  in  the  annexed 

«7  Dippel,  I.e.,  p.  131. 

«  Moller  in  Wedel,  Holstein,  offers  such  at  the  price  of  1.50  Marks. 


70 


THE  MICROSCOPE- IN   BOTANY. 


table  the  results  arrived  at  by  this  well-known  naturalist.49 
(Cross  markings  are  always  meant  except  in  Surirella  where 
longitudinal  lines  are  intended.  In  Ijycaena,  a  signifies  a 
bright  colored  and  b  a  dark  colored  scale.) 


NAME  OF  TEST  OBJECT. 

Manner 
of 
Mounting. 

Number  of 
strife  to 
the  0.01  mm. 

Distance  apart 
of  the  strice 
in  Millimeters. 

Balsam 

4—6 

0.00208 

7—8 

0.00153 

Drv 

10        12 

0  00099 

10  —  11 

0  00096 

«               «                     6  

H 

14  —  15 

0  00074 

1  l.'l  ]-.'i  III 

14        15 

0  00074 

Drv 

22        23 

0.00046 

25 

0  00041 

<( 

28  —  29 

0.00036 

Dry 

30  —  32 

0.00032 

32        34 

0.00031 

Applying  the  tests  in  the  series  in  accordance  with  the  ar- 
rangement in  this  table,  one  can  come  to  a  clear  understanding 
of  the  excellency  and  of  the  working  qualities  of  his  instrument. 

In  conclusion  it  may  be  mentioned  that  Nobert  for  a  long 
time  has  been  making  and  furnishing  an  apparatus  for  testing 
the  microscope  without  the  use  of  test  objects.  It  is  called 
"Nobert's  test  plate"  (Probeplatte).  It  consists  of  a  number 
of  groups  of  very  delicate  lines  which  are  cut  in  glass  by  means 
of  a  diamond,  or  eaten  into  it  by  means  of  hydrofluoric  acid. 
The  lines  of  the  several  groups  are  at  different  distances  apart, 
as  for  example,  those  of  the  first  are  0.002256  mm.  and  of  the 
last  group,  0.000282  mm.  asunder.  The  distance  apart  of  the 
lines  of  the  groups  lying  between  these,  gradually  pass  in  value 
from  that  of  the  former  to  that  of  the  latter.  It  is  evident  that 
with  the  help  of  this  plate  one  may  very  easily  determine  the 
resolving  power  of  a  system,  if  he  will  begin  with  the  first  group 
and  work  progressively  through  toward  the  more  difficult  till 
he  has  reached  a  point  where  his  lens  will  no  longer  resolve  the 
lines.  The  Nobert  test  plate  would  certainly  supersede  all 

«  Dippel,  1.  c.,  p.  134/. 


THE  MICROSCOPE  TUBE.  71 

other  test  objects,  did  not  its  very  high  price,  made  necessary 
by  the  almost  incredible  fineness  of  the  work,  interpose  the 
greatest  barrier  to  its  general  distribution.* 

Since  we  have  now  finished  the  consideration  of  the  optical 
parts  of  the  microscope,  we  will  undertake  to  describe  the  other 
parts,  only  however  in  respect  to  their  principal  features,  the 
stand  and  the  illuminating  apparatus.  We  will  first  describe 
the 'microscope-tube. 

VIII.     THE  MICROSCOPE-TUBE. 

The  microscope-tube  is  a  solid  tube  of  brass,  whose  length 
is  adjusted  to  the  construction  of  the  optical  apparatus  and  rel- 
atively to  the  cooperation  of  the  objective  and  ocular.  In  the 
medium  and  larger  microscopes,  its  length  varies  between  18 
and  28  cm.  Beneath,  it  carries  the  ["Society"]  screw  for  re- 
ceiving the  objective-system.  Within  its  upper  opening  which 
is  made  cylindrical  to  receive  it,  the  ocular  is  set.  Within  the 
tube  are  placed  several  diaphragms  for  cutting  off  certain  rays 
which  would  injure  the  microscopic  image.  The  tube  is 
blackened  on  the  inside,  at  least  in  the  lower  part  and  up  to  the 
topmost  diaphragm.  [In  some  instruments,  lining  the  inner  tube 
'with  lusterless  black  cloth  will  improve  the  definition  by  remov- 
ing a  scarcely  noticed  glare  of  light  reflected  from  the  imper- 
fectly deadened  surface  of  the  brass  tube.  In  other  stands, 
diffused  light  enters  the  short  oculars  from  the  adjacent  surface 
of  the  tube  that  has  been  brightened  by  contact  with  the  longer 
oculars  :  this  is  prevented,  by  some  makers,  by  so  shaping  the 
oculars  or  so  guarding  the  inside  of  the  tube  that  all  oculars, 
whatever  their  length,  will  come  into  contact  with  the  tube  for 
exactly  the  same  distance.  R.  H.  W.] 

The  larger  microscopes  commonly  have  draw-tubes  also.  The 
tube  then  consists  of  two  parts,  one  shoving  into  the  other  like 
a  telescope,  so  that  it  may  be  drawn  out  to  different  lengths, 
according  to  need.  On  the  advantage  of  this  arrangement, 
Harting  has  remarked  as  follows  i50 

50  Harting.  Mikr.,  page  157/, 

*Ou  account  of  the  recent  death  of  Xobert.  his  rulings  will  become  increasingly 
scarce;  but  plates  approaching  them  in  quality,  and  serving  the  same  purpose  as  tests,  are 
now  ruled  at  a  far  less  cost  by  C.  Fasoldt  of  Albany,  N.Y.,  and  by  other  makers.— R.  H.  W. 


72  THE  MICROSCOPE  IN  BOTANY. 

"In  a  microscope  to  which  belong  several  oculars  and  objec- 
tives, it  would  be  unreasonable  to  expect  that  one  and  the  same 
length  of  tube  would  be  best  for  all  combinations.  An  exam- 
ination previously  made  will  serve  to  show  at  what  length  of 
tube  the  various  optical  parts  will  do  their  best  work,  and  this 
can  be  noted  and  followed  in  the  future." 

"A  second  advantage  of  this  contrivance  consists  in  this,  that 
by  the  inward  and  outward  movement  of  the  inner  tube,  the 
magnification  can  be  brought  to  any  definite  number.  For  mak- 
ing micrometric  measurements  this  is  very  important.  It  is 
simpler  for  instance  to  have  the  diameter  of  the  image  divided 
by  500  than  by  487  or  513,  or  by  100  than  by  93  or  107.  Also 
in  many  observations,  it  is  important  to  have  the  field  of  view 
of  a  certain  definite  size,  1,  2,  3,  etc.,  mm.  But  this  can  be 
brought  about  only  by  increasing  or  diminishing  the  distance 
between  the  ocular  and  the  objective,  and  this  may  be  done  with- 
out damage  to  the  image  if  certain  limits  be  not  overstepped." 

"For  this  purpose  the  inner  tube  should  be  graduated.  The 
optician,  or  the  owner  of  the  microscope,  can  then,  by  careful 
investigation,  construct  a  table  which  shall  indicate  the  point 
on  the  graduation  which  will  be  of  service  in  actual  work  with 
various  combinations  and  magnifications." 

[A  third  use  of  the  draw-tube  is,  by  being  pushed  in,  to  re- 
duce the  length  of  the  body  to  much  less  than  its  usual  standard, 
for  the  purpose  of  adapting  it  to  the  vertical  position  often  re- 
quired in  laboratory  work.  To  this  end,  such  stands  as  are  most 
suitable  for  histological  work  are  made  with  a  very  short  body, 
about  12  cm.  long,  which  may  be  increased  to  the  ordinary  length 
when  using  the  instrument  in  an  inclined  position,  by  extending 
the  draw-tube  as  represented  in  Plate  III.  In  Plate  X  the  draw- 
tube  is  wholly  closed.  The  stand  shown  in  Plate  XI  has  two 
draw-tubes,  one  within  the  other,  to  give  greater  range  of  length. 
By  this  extreme  shortening  of  the  body,  the  ocular  is  brought 
as  near  as  possible  to  the  table  and  one  is  enabled,  with  a  min- 
imum of  discomfort  and  fatigue,  to  lean  over  the  stand  and  look 
down  the  vertical  tube.  While  the  optical  corrections  are  visibly 
disturbed  by  great  shortening  of  the  tube,  there  are  many  ob- 
jectives of  moderate  capacity  whose  performance  is  not  materially 


THE  DRAW-TUBE.  73 

injured,  and  others  whose  screw  collar  adjustment  is  capable  of 
fairly  correcting  the  evil  produced.] 

[The  draw-tube  usually  is,  and  should  in  all  cases  be,  provided 
at  its  lower  end  with  an  adapter  having  the  society  screw  for 
the  reception,  when  required,  of  an  analyzing  prism,  a  spectro- 
scopic  arrangement  or  an  objective  to  serve  as  an  erector.  An 
objective,  having  too  great  focal  length  to  be  employed  in  the 
usual  manner,  may  likewise  be  inserted  here  and,  by  sliding 
the  draw-tube,  it  may  be  focussed  through  the  empty  nose-piece 
upon  the  object  below  it.  Should  the  screw  become  bright  from 
use  and  reflect  false  light,  it  must  be  guarded  by  a  ring  of  hard 
rubber  or  blackened  brass  screwed  into  it.  This  will  not  only 
render  it  harmless  but  will  become  an  efficient  diaphragm  to 
stop  stray  light  from  other  sources.  R.  H.  W.] 

The  microscope-tube  is  moved  by  a  propelling  mechanism, 
rack  and  pinion,  or  by  free  hand.  In  the  latter  case  it  is  nec- 
essary that  the  tube  should  exactly  fit  into  the  inclosing  sheath. 
This  is  secured  by  careful  polishing  of  both  surfaces  or  by  lining 
the  inside  of  the  sheath  with  cloth  [or  by  the  use  of  springs]. 
The  tube  should  never  be  oiled,  but  it  and  the  sheath  kept 
always  absolutely  clean. 

When  we  are  examining  one  object  after  another  with  different 
magnifying  powers,  it  is  necessary  in  each  case  to  screw  another 
system  into  the  tube. 

[The  screw  by  which  the  objectives  are  attached  to  the  tube 
is  of  standard  size,  devised  and  prepared,  in  1857,  by  the  Lou- 
don  (now  Royal)  Microscopical  Society,  and  therefore  known 
as  the  "  Society"  screw.  Being,  practically,  in  universal  use 
in  both  this  country  and  England,  and  even  applied  to  conti- 
nental objectives  intended  for  sale  here,  this  standard  screw 
renders  objectives  of  the  different  makers  interchangeable,  so 
that  a  student  may  use  his  set  of  objectives  upon  a  variety  of 
stands,  or  may  purchase  any  desired  lens  without  doubt  as  to 
its  harmonizing  with  his  former  apparatus.] 

[The  introduction  of  the  society  screw,  with  all  its  attendant 
advantages,  was  a  loss  in  respect  to  ease  of  manipulation,  as  it 
put  a  stop,  temporarily,  to  the  use  of  bayonet  catches  and  other 
devices  designed  to  lessen  the  labor  and  delay  occasioned  by 
frequent  changes  of  objectives  while  the  instrument  was  in  use.] 


74  THE  MICROSCOPE   IN   BOTANY. 

[IX.    NOSE-PIECES.] 

[Subsequently  double,  and  even  triple  and  quadruple  nose- 
pieces  were  used,  the  upper  portion  of  the  apparatus  being 
attached  by  a  society  screw  to  the  compound  body,  while 
the  lower  portion  or  revolving  plate  carried  the  specified 

number  of  objectives  permanently 
screwed  into  it,  so  that  any  one 
of  them  could  be  rotated  into  use, 
in  the  axis  of  the  instrument.  Such 
a  triple  nose-piece  is  shown  in  Fig. 
18.  When  well  made,  very  light, 
and  of  the  angular  form,  this  device 
is  very  satisfactory  in  use,  and 
greatly  relieves  the  labor  of  inves- 
tigations requiring  frequent  changes 

of  power.  It  is  however  somewhat  costly  and  cumbersome, 
and  its  weight,  including  that  of  the  attached  objectives,  im- 
pairs the  delicacy  of  the  fine  adjustment,  especially  in  some  of 
its  older  forms.] 

[During  the  past  two  or  three  years  there  has  come  into  ex- 
istence a  new  series  of  contrivances,  by  which  objectives  though 
attached  one  at  a  time  can  be  changed  with  ease.  In  them  the 
nose-piece  is  a  single  chuck,  permanently  screwed  into  the  mi- 
croscope body,  some  mechanism  being  included  by  which  the 
objective  can  be  instantly  seized  or  released.  No  change  is 
made  in  the  screw  of  the  microscope-tube  or  of  the  objective, 
and  the  collar  screwed  upon  the  objective  should  be  of  such  size 
as  to  be  left  in  position  when  the  objective  is  packed  in  its  box. 
These  nose-pieces  require  excellent  workmanship,  in  order  to 
secure  accurate  centering  and  adequate  stability,  and  in  their 
use  care  should  be  exercised,  especially  by  inexpert  hands  dur- 
ing the  manipulations  required  for  the  sere w-collar  adjustment 
of  the  objective,  not  to  unintentionally  release  the  objective 
from  the  grasp  of  the  chuck.  Should  experience  give  weight 
to  this  drawback,  slight  modifications  of  the  apparatus  would 
doubtless  remove  the  difficulty.*] 

,  *  Constant  use  of  the  "Facility"  nose-piece  for  more  than  a  year  does  not  reveal  the 
existence  of  this  possible  objection.— A.  B.  H. 


NOSE-PIECES. 


75 


FIG.  J9. 


[Such  a  contrivance  is    the  "Facility  "  nose-piece,  Fig.  19, 
which  is  simply  a  self-centering  chuck,  which  seizes  the  objec- 
tive by  a  small  ring  or  collar  permanently  screwed  upon  its  so- 
ciety   screw.      It    was    devised   and  is 
made  by  James  L.  Pease  of  Chicopee, 
Mass.      Of  the    same  general  character 
is  the  "  Congress"  nose-piece  contrived 
by  Prof.  Albert  McCalla  and  made  by 
W.  H.  Bulloch  of  Chicago,  Fig.  20,  in 
which  a  chuck,  with  three  slots,  grasps, 
by    three    pro- 
jections upon  it, 
a    ring    perma- 
nently   screwed 

upon  the  objective.  The  contrivance 
still  more  recently  produced  by  Mr. 
Zentmayer,  Fig.  21,  is  one  in  which 
a  nose-piece  is  screwed  into  the  micros- 
cope-tube and  a  collar  permanently 
screwed  upon  the  objective,  the  collar 
and  nose-piece  being  connected  by  a 
screw,  the  opposite  quarters  of  whose 
threads  are  cut  away,  so  that  insertion 
can  be  accomplished  without  screwing 
and  the  inserted 
collar  locked  fast 
by  a  single  quarter- 
turn  which  will  bring  the  screw-threads  of 
the  two  pieces  into  relation  with  each  other. 
An  index-mark  upon  the  nose  and  a  corre- 
sponding mark  upon  each  collar  indicate  the 
position  in  which  the  collar  can  be  inserted  ; 
and  a  jam-nut  enables  the  nose  to  be  set  in 
such  position  as  to  bring  its  index  in  front 
of  the  microscope  or  in  any  location  pre- 
ferred by  the  observer.  This  contrivance 
has  been  successfully  used  in  the  arts,  as  in  the  construction 
of  breech-loading  cannon  and  in  the  coupling  of  hose  for  use 


FIG.  20. 


FIG.  21. 


76  THE   MICROSCOPE   IN  BOTANY. 

with  "  fire-engines."  It  was  proposed  by  Mr.  E.  M.  Nelson 
at  the  Qtieckett  Club,  Sept.,  1882,  to  similarly  cut  away 
portions  of  the  society-screw  from  objectives  and  stands,  as 
a  means  of  instantaneous  attachment.  No  general  effort  has 
been  made,  however,  to  introduce  it  for  this  purpose,  and  it  is 
doubtful  if  the  modification  could  be  effected  by  the  various 
makers  with  such  uniformity  that  their  work  would  be  really 
interchangeable.] 

[The  latest  contrivance  of  this  sort  is  by  Mr.  Charles  Fasoldt 
of  Albany,  N.  Y.,  who  makes  a  nose-piece  the  alternate 
sixths  of  which  have  the  thread  cut  away,  while  one  of  the 
remaining  sixths  is  movable  and  capable  of  being  withdrawn 
from  contact  by  a  lever  reacting  against  a  strong  spring,  as 
shown  in  Fig.  22.  The  objective  requires  no  preparation,  and 

can,  after  starting  by  pushing 
back  the  movable  section  of 
the  screw,  be  screwed  in  in 
the  usual  manner.  If,  how- 
ever, the  lever  be  pressed  down 
with  the  thumb,  the  movable 
section  of  the  screw  is  with- 
drawn so  far  that  the  objec- 
FIG  2£>  tive  can  be  placed  at  once  in 

position  against  the  shoulder 

above.  Releasing  the  lever  allows  the  movable  portion  of  the 
screw  to  slide  firmly  back  into  place  and  grasp  the  threads 
of  the  objective,  which  is  held  exactly  as  if  it  had  been  screwed 
in,  and  which  can  be  tightened  up  against  the  shoulder,  if  neces- 
sary, by  a  single  screwing  movement.  A  jam-nut  is  provided, 
as  in  the  Zentmayer  form,  by  which  the  lever  and  index  can  be 
set  in  a  position  most  convenient  to  the  observer.  A  correspond- 
ing mark  should  be  made  upon  the  brass  mounting  of  every 
objective  to  be  used,  the  mark  upon  the  objective  corresponding 
to  the  index  of  the  nose-piece,  not  when  the  objective  is  snugly 
screwed  up  to  the  proper  tension,  but  after  it  has  been  un- 
screwed about  one-eighth  of  a  revolution.  When  then  in- 
serted, in  this  slightly  unscrewed  position,  the  threads  are 
grasped  with  more  certainty  and  effect,  and  a  slight  twist,  one- 


THE  FINE-ADJUSTMENT.  77 


eighth  turn  to  the  left,  sets  it  with  uniform  and  unerring  firin- 
iiess  against  the  shoulder.  After  experience  renders  this  twist 
habitual,  it  becomes  almost  automatic,  and  is  scarcely  distin- 
guished as  a  part  of  the  apparently  single  action  of  putting  the 
objective  where  it  is  wanted.  When  in  position,  the  objective 
cannot  fall  out  or  be  pulled  out,  without  being  either  unscrewed 
or  else  instantly  released  by  pressing  down  the  lever  that  opens 
the  screw.  R.  H.  W.] 


X.     THE  FINE-ADJUSTMENT. 

The  fine  adjustment  screw,  as  we  have  pointed  out,  is  a  con- 
trivance by  which,  when  the  coarse  adjustment  has  brought  the 
optical  apparatus  into  the  immediate  neighborhood  of  the  ob- 
ject, it  can  be  adjusted  with  almost  mathematical  exactness,  so 
as  to  bring  the  object  exactly  into  the  focal  point  of  the  lens. 
How  this  adjustment  was  brought  about  in  the  older  micro- 
scopes we  have  already  mentioned,  p.  7.  With  smaller  micro- 
scopes, to  this  day,  the  fine  adjustment  is  occasionally  found  on 
the  stage.  This  is  objectionable  on  instruments  used  for  sci- 
,  entific  observation.  In  microscopes  designed  for  scientific 
investigation  the  fine-adjustment  screw  is  placed  in  the  perpen- 
dicular pillar  [or  variously  shaped  limb]  which  bears  the 
tube. 

[Until  a  very  few  years  ago,  the  fine  adjustment  of  the  best 
English  and  American  stands  consisted  of  a  light  tube  or  nose- 
piece,  sliding  easily  within  the  main  body  of  the  microscope 
and  projecting  slightly  below  its  lower  end ;  this  tube  being 
pressed  firmly  downward  by  a  spiral  spring,  and  being  raised 
by  a  lever,  actuated  by  a  screw  which  was  turned  by  the  thumb 
and  fingers  of  the  observer's  hand.  The  position  of  the  lever 
and  screw  varied  with  the  caprice  of  the  maker,  but  it  was 
most  frequently  attached  to  the  body  in  front  and  near  the  lower 
end.  The  objective  screwed  into  this  sliding  tube  could  be 
moved  up  and  down  until  its  distance  from  the  object  was  ad- 
justed with  great  precision.  The  arrangement,  however, 
lacked  firmness,  especially  in  handling  the  screw-collar  adjust- 


78  THE  MICROSCOPE  IN  BOTANY. 

ment,  and  it  often  became  much  the  worse  for  wear.  Some 
persons,  also,  feared  that  the  slight  change  it  effected  in  the  dis- 
tance between  the  objective  and  the  ocular  might  be  practically 
as  well  as  theoretically  objectionable.] 

[Meanwhile  Continental  microscopes  were  made  with  a  fine 
adjustment  moving  the  whole  body  of  the  instrument.  In 
most  of  those  which  found  their  way  to  this  country  or  were 
imitated  here,  a  hollow  pillar  was  provided,  within  which  was 
contained  a  solid  cylinder  firmly  attached  below  to  the  foot,  or 
to  the  movable  portion  of  the  trunnion-joint.  The  outer  por- 
tion (a  hollow  tube  sliding  over  the  inner)  carried  with  it  by 
means  of  a  transverse  bar  the  whole  body  of  the  microscope, 
the  vertical  movement  being  accomplished  by  a  screw  working 
against  a  spring  ;  just  as  if,  in  Plate  XI,  the  milled  screw-head, 
high  up  at  the  right  of  the  plate,  were  made  (which  it  is  not)  to 
carry  up  and  down  the  outer  and  visible  portion  of  the  column 
below  it  over  an  inner  and  concealed  column.  This  form  of 
adjustment  lacked  smoothness  and  uniformity  of  movement, 
was  particularly  subject  to  side  motion,  and  had  no  good  means 
of  taking  up  the  loss  from  wear.  Its  use  in  this  country  was 
very  limited  and  mostly  confined  to  stands  of  low  grade.] 

[In  1876,  among  the  novelties  prepared  for  display  at  the 
Centennial  exhibition,  Mr.  Zentmayer  transferred  the  fine  ad- 
justment of  his  stand  from  the  nose-piece  to  the  limb,  making 
the  bar  just  behind  the  body  slide  upon  plane  surfaces,  as  in 
the  coarse  adjustment,  a  sufficiently  delicate  movement  being 
imparted  to  it  by  a  lever  acted  upon  by  a  screw  at  the  left. 
Great  steadiness  and  indefinite  capacity  for  wear  are  attained  in 
this  way.  This  adjustment  is  shown  in  Plate  III,  the  black 
line  just  back  of  the  body  and  parallel  with  it  representing  the 
edge  of  the  sliding  surfaces.  In  Plate  IX  this  is  shown  com- 
bined with  a  rack  and  pinion  coarse  adjustment,  a  separate  slid- 
ing movement  being  provided  for  each.] 

[In  Mr.  Bulloch's  microscopes,  Plate  X,  a  similarly  situated 
screw  and  lever  give  a  very  delicate  vertical  motion  to  a  sliding 
box,  in  which  is  set  the  pinion  of  the  coarse  adjustment  itself 
as  a  portion  of  the  fine  adjustment.] 

[In  the  Bausch  and  Lomb  instruments  may  be  found  the  so- 


THE  FINE-ADJUSTMENT. 


79 


called  "clock-spring"  fine  adjustment,  shown  in  Plates  IV  and 
XI,  and  in   section    in  Fig.    23.      The  transverse  bar  of  the 
stand,  extending  from  the  pillar  to  the  body,  consists  of  a  hollow 
box  whose  top  is  represented  by  d,  whose  front,  towards  the  right, 
is  open,  and  whose  back,  to- 
wards the  left,  is  either  at- 
tached  to    the   pillar   c   by 
screws,  or  cast  in  one  piece 
with  it,  as  in  Plate  IV.  From 
the  back  of  this    box,    and 
firmly  attached  to  it,  project 
forward  two  stiff  horizontal 
steel  springs  aa,  which  bear 
at  the  right  the  plate  e,  con- 
taining the  pinion  f  of  the 
coarse  adjustment  fg.      An 
arm  of  e  projects  backwards 
and  is  pressed  down  by  the 
carefully  cut  fine-adjustment 
screw  6,  reaction  being  fur- 
nished   by   the    springs  aa. 
The  horizontal  and  vertical 
portions  of  e  being  inflexible 
and     continuous,     and     the 
pinion-rack  and  body,  f  to 
A,  having  no  other  support 
than  the  springs  aa,  it  is  evident  that  any  vertical  movement 
of  the  screw  b   must  impart   a   like    movement   to  the    body 
h    and    to    the    optical  parts  contained  therein.     Since  a  and 
a    are  parallel  and  of  equal  length,  the  motion  of  the  body 
h  must  always  be  in  a  direction  absolutely  parallel  to  the  pillar 
c  and  vertical  to  the  plane  of  the  stage.     The  theoretical  move- 
ment of  h  to  and  from  c  is  so  slight  as  to  be  unnoticed.     This 
form  of  adjustment   is  free  from  lateral  motion  or  lost  motion, 
has  no  friction  except  that  of  the  screw  itself  and  does  not  de- 
teriorate by  age  or  wear.     A  somewhat  similar  system,  though 
not  exempt  from  friction  and  wear,  is  employed  in  the  stands  of 
Seibert  and  Krafft  described  by  Dr.  Behrens,  in  which  a  par- 


FlG.  23. 


80  THE  MICROSCOPE  IN  BOTANY. 

allel  motion  is  secured  by  a  system  of  levers  instead  of  springs. 
R.  H.  W.] 

XL     THE  STAGE. 

The  object-table,  or,  for  short,  the  stage,  is  a  solidly  wrought 
metal  plate  which,  in  respect  to  the  optical  apparatus,  assumes 
an  unalterable  position.  The  optical  axis  of  the  microscope 
must  be  exactly  perpendicular  to  the  plane  of  the  stage.  The 
form  of  the  stage  as  in  Plate  X  is  rectangular,  or,  as  in  Plate  XI, 
round.  Both  forms  are  alike  practical,  assuming  that  they  are 
roomy  enough  to  be  convenient  to  handle.  Small  stages  are 
objectionable.  They  should  at  least  be  large  enough  so  that  the 
largest  form  of  slide  would  not  reach  from  side  to  side.  In 
order  to  obviate  the  reflection  of  rays  from  the  stage  it  is  com- 
monly blackened  [at  least  on  its  lower  side]. 

Large  microscopes  are  usually  furnished  with  rotating  stages, 
and  this  arrangement  is  very  convenient,  as  it  enables  one  to 
turn  the  object  quite  about  upon  its  axis  without  being  obliged 
to  disturb  the  slide.  And  besides,  when  the  rotating  stage  is 
graduated  and  made  to  work  by  an  index,  it  can  be  used  with 
good  results  in  measuring  the  angles  of  crystals.  In  the  latter 
case,  the  stage  should  be  provided  with  a  screw  arrangement 
by  which  it  may  be  exactly  centered,  so  as  to  bring  that  part 
of  the  object  to  be  examined  into  the  exact  optical  axis  of  the 
microscope.  In  the  illustrations,  Plates  X  and  XI,  such  ro- 
tating stages  are  represented  [centering  adjustment  being  ap- 
plicable if  specially  ordered].  The  circular  plate  can  be  set  in 
rotary  motion  upon  the  stationary  tinder-piece.  It  has  a  milled 
edge  so  as  to  be  moved  more  easily  by  hand. 

[Many  of  the  small  and  cheap  microscopes  are  now  made 
with  a  plain  round  stage,  a  form  unobjectionable  in  itself  and 
capable  of  a  higher  development  than  the  square.  The  stage, 
for  instance,  in  Plates  IV,  V  or  VI,  is  of  extreme  simplicity, 
but  is  capable,  by  reason  of  its  circular  form  and  location  (con- 
centric with  the  optical  axis),  of  receiving,  either  originally  or 
subsequently,  such  an  upper  plate  as  those  shown  in  Plates  X 
and  XI,  thus  making  a  simple  but  serviceable  revolving  stage. 


THE  STAGE.  81 

Such  ail  arrangement,  besides  its  other  good  qualities,  is  com- 
patible with  that  extreme  thinness  of  stage  which  is  now  con- 
sidered essential  in  order  that  freedom  of  illumination  be  not 
interfered  with.  R.  H.  W.] 

It  is  sometimes  necessary  to  make  the  object  fast  to  the  stage 
in  order  to  devote  a  considerable  time  to  the  examination  of  a 
single  point,  or  in  order  to  be  able  to  turn  the  microscope  down 
to  an  oblique  position.  For  this  purpose  a  simple  clamp  ar- 
rangement is  sufficient. 

[The  commonest  form,  and  one  answering,  in  skilful  hands, 
nearly  all  useful  purposes,  is  a  pair  of  spring  clips  of  steel  or 
brass  attached  to  the  stage  by  screws  or  pins,  as  shown  in  Plates 
III  and  XI.  Beneath  these  the  object-slide  is  placed  and  can 
be  successfully  manipulated,  even  under  moderately  high  powers. 
A  bar  of  glass  or  brass,  sliding  under  short  clips,  forms  a  suffi- 
cient ledge  for  the  support  of  a 
trough  or  receptacle  too  large 
to  be  placed  under  the  clips. 
A  some  what  more  delicate  adjust- 
ment can  be  obtained  by  using 
a  brass  object-carrier  sliding 

over  a  glass  stage  as  in  Fig.  24,  which  is  designed  as  an  addition 
to  the  stand  shown  in  Plate  IV ;  or  a  glass  object-carrier,  or 
sliding  stage,  which  is  especially  adapted  to  chemical  work  and 
is  easily  applied  to  any  stage  of  the  model  shown  in  Plate  IX.] 

[While  elaborate  "  mechanical  stages,"  on  which  the  object 
can  be  moved  in  every  direction  by  racks  and  screws,  are  not 
required  for  general  use  and  are  much  less  esteemed  and  used 
than  formerly,  still  there  are  some  procedures,  such  as  microm^ 
etry  and  the  use  of  extremely  high  powers,  where  a  stage  having 
some  sort  of  mechanical  movement  is  a  material  advantage. 

o 

Such  stages  are  now  made  in  simplified  forms,  thin  enough  to 
conform  to  the  present  demand  for  thin  stages,  and  small  enough 
to  be  applicable  to  the  smallest  stands  described  in  this  book. 
R.  H.  W.] 

An  arrangement  seldom  employed  by  the  botanist,  but  more 
frequently  used  by  the  zoologist,  is  a  warming  stage.     By  means 
of  it,  the  preparation  may  be  kept  at  a  higher  temperature  than 
6 


82  THE    MICROSCOPE   IN  BOTANY. 

that  of  the  surrounding  atmosphere.  Max  Schulze51  has  invented 
such  a  stage  capable  of  being  heated.  It  consists  of  a  metal 
plate  which  is  fastened  to  the  stage  by  clamps.  Corresponding 
to  the  opening  in  the  stage  there  is  a  place  bored  out  in  it  for 
illumination.  It  carries  in  front,  in  the  middle  a  diagonally 
placed  thermometer  and  two  long  arms  extend  out  far  beyond 
the  stage ;  under  these  are  placed  two  spirit  lamps  as  heaters. 
The  lower  end  of  the  thermometer  is  wound  about  the  opening 
on  the  plate  and  so  gives  the  exact  temperature  of  the  object. 


XII.     THE  ILLUMINATING  APPARATUS. 

The  illuminating  apparatus  consists  essentially  of  three  in- 
struments, viz.  :  the  mirror,  the  diaphragm  and  the  condensing 
lens.  The  mirror  is  placed  beneath  the  stage,  but  the  diaphragm 
.is  placed  near,  if  not  upon  the  under  side  of  the  stage  itself. 


A.    THE  MIRROR. 

The  mirror  is  formed  of  a  circular  metal  frame  of  30  to  50  mm. 
[or  more]  broad,  in  which  is  mounted,  on  one  side,  commonly, 
a  plane  glass  mirror,  and  on  the  other,  a  concave  mirror  of 
spherical  form.  [The  mirror  is  fastened  to  the  arm,  as  seen  in 
Plates  III  and  XL]  The  mirror  frame  is  mounted  upon  an 
arm  so  as  to  revolve  upon  it  and  the  arm  is  so  constructed  as 
to  enable  the  mirror  to  be  moved  right  and  left,  and  placed  in 
any  position  with  reference  to  the  opening  in  the  stage. 

It  is  by  no  means  a  matter  of  indifference,  for  the  illumination 
of  the  object,  which  of  the  two  mirrors,  the  plane  or  the  concave, 
is  used,  as  will  be  clear  from  what  follows.  We  will  suppose 
that  there  is  a  source  of  light  I,  Fig.  25,  which  sends  rays  upon 
the  mirror  s,  as  shown  in  the  illustration,  Za,  Ib,  Ic,  Id,  le.  It 
is  known  that  the  concave  mirror  converges  the  rays  that  it 
reflects.  It  will  be  seen  without  further  elucidation  that,  by 
giving  the  mirror  its  proper  position  and  distance  from  the 

6i  Frey,  Das  Mikroskop,  page  65. 


THE  MIRROR.  83 

object,  all  the  rays  which  fall  upon  it  will  be  concentrated  upon 
the  object  that  lies  at  p,  upon  the  stage  between  the  slide  and 
the  cover-glass.  In  this  case  very  many  rays  are  concentrated 
upon  a  very  small  space,  the  object,  which  must  consequently 
be  very  brightly  illuminated.  The  parallel  rays  of  daylight, 
which  are  commonly  used  in  microscopical  investigations,  behave, 
when  they  fall  upon  the  concave  mirror  quite  like  the  diverging 
rays  we  have  just  been  considering. 

The  plane  mirror  works  very  differently.  The  parallel  rays 
falling  upon  that  are  reflected  at  the  same  angle,  consequently 
run  parallel  on  their  way  to  the  object.  If  we  now  suppose 


FIG.  25. 

that  there  falls  upon  the  surface  of  the  mirror  n  rays  of  light 
and  the  stage  diaphragm  has  but  L  the  extent  of  the  mirror 
surface,  it  follows  that  the  plane  mirror  will  afford  but  JB.  of  the 
light,  to  the  object,  that  it  receives,  while  under  the  same  con- 
ditions, the  concave  mirror  would  contribute  to.  the  illumination 
of  the  object,  all  of  the  rays  that  it  receives.  Still  less  of  the 
illumination  will  be  given  to  the  object  when  it  is  received  from 
a  near  source  of  light  as  in  Fig.  25,  for  then  the  rays  are  di- 
verging, and  on  falling  upon  the  plane  surface,  will  be  still  more 
diverged,  and  thus  spread  more  widely,  and,  so  to  say,  thinly, 
over  a  given  space. 

For  obvious  reasons,  therefore,  the  plane  mirror  should  be 
used  with  low  powers,  and  the  concave  with  higher  and  the 


84  THE  MICROSCOPE  IN  BOTANY. 

highest  powers.  From  the  above  considerations  also,  it  is  easily 
seen  why  the  plane  mirror  affords,  on  the  whole,  stronger  lines 
of  definition  than  the  concave.  Very  delicate  structures,  which 
are  to  be  observed  with  low  powers,  appear  with  much  sharper 
outlines  when  illuminated  by  the  plane  than  by  the  concave  mirror. 
For  many  objects,  with  medium  or  high  magnification,  illumi- 
nation must  be  had  from  the  concave  mirror  with  oblique  light, 
in  order  to  be  able  to  recognize  certain  fine  details.  This  oblique 
illumination  is  produced  by  moving  the  mirror  to  a  certain  angle 
right  or  left,  and  adjusting  the  diaphragm  so  that  the  cone  of 
rays  may  still  reach  the  object  on  the  stage.  Now  the  outlines 
will  give  broader  shadows  than  before  and  so  will  be  much  more 
easily  recognized.  The  different  effects  of  central  and  oblique 
illumination  become  clear  by  using  first  one  and  then  the  other 
illumination  upon  the  diatoms,  as,  for  example,  Pleurosigma 
angulatum,  PL  formosum  or  PL  balticum. 

B.     DIAPHRAGMS. 

Only  in  rare  cases  will  one  be  able  to  obtain  the  desired 
illumination  by  means  of  the  mirror  alone.  In  many  cases  it 
is  important  to  shut  off  from  the  microscopic  image  the  border 

rays  and  even  the  central  rays  that 
are  reflected  from  the  mirror. 
Both  can  be  accomplished  by 
means  of  the  diaphragm  attached 
to  the  stage. 

1 .  The  Revolving  Diaphragm. 
In  the  older  instruments  and  in 
the  smaller  ones  of  the  present 
time  the  marginal  rays  are  shut 
off  by  means  of  the  revolving 
diaphragm,  Fig.  26.  It  is  a 
FIG-26-  plate  of  metal,  not  too  thick, 

blackened  on  both  sides  and  provided  with  several  round  holes 
of  different  diameters,  but  whose  middle  points  are  at  the  same 
distance  from  the  center  of  the  disk.  The  diaphragm  is  fastened 
to  the  under  side  of  the  stage  [or  to  some  supporting  apparatus 


DIAPHRAGMS.  85 

beneath  the  stage]  by  a  screw  passing  through  its  center,  and 
about  which  it  is  made  to  turn  by  the  fingers,  its  openings  being 
brought  successively  under  the  opening  in  the  center  of  the 
stage. 

But  it  is  difficult  faultlessly  to  center  this  diaphragm,  and 
what  is  a  worse  evil  it  is  not  on  the  same  plane  with  the  top  of 
the  stage  but  with  the  under  surface,  and  the  consequence  is 
that  between  the  diaphragm  and  the  object  a  cone  of  dispersing 
rays  is  formed  which  very  considerably  injures  the  clearness  of 
the  microscopic  image.  To  avoid  this  fault  the  .diaphragm  is 
sometimes  made  in  the  form  of  a  concave  segment  of  a  globe,  and 
the  under  side  of  the  stage  is  hollowed  out  to  correspond.  But 
this  contrivance  allows  no  very  near  approach  to  the  object,  and 
the  difficulty  of  correct  centering  is  still  greater,  and  the  objec- 
tion to  this  kind  of  diaphragm  increases  with  the  power  of  the 
objective  employed  in  the  investigation. 

[In  a  few  American  stands,  as  in  some  of  those  made  by  Mr. 
Grunow  of  New  York,  and  in  one  of  Mr.  Zentmayer's,  the  re- 
volving diaphragm  plate  is,  for  this  reason,  let  into  the  upper  sur- 
face of  the  stage,  so  that  when  in  use  it  lies  almost  in  contact  with 
the  object-slide.  In  most  American  microscopes,  however,  it  is 
attached  to  the  under  surface  of  the  stage  or  fixed  at  a  moder- 
ate distance  below  it,  in  order  to  secure  that  control  of  the  an- 
gular breadth  of  the  illuminating  pencil,  which  is  obtained  by 
locating  the  diaphragm  at  a  sensible  distance  below  the  point 
of  convergence,  p,  in  Fig.  25,  of  the  cone  of  rays  condensed  up- 
on the  object  by  the  concave  mirror.  By  far  the  best  arrange- 
ment is  to  have  the  diaphragn^-plate  so  mounted,  whether  in  a 
sliding-tube  or  upon  a  sub-stage,  that  it  can  be  set  at  any  level 
from  the  plane  of  the  top  of  the  stage  to  a  plane  five  or  ten 
mm.  below  it,  as  in  Plates  IX  to  XI.  R.  H.  W.] 

2.  The  Cylindrical  Diaphragm  is  a  contrivance  which  quite 
obviates  the  evils  mentioned,  as  belonging  to  the  disk  dia- 
phragm, and  it  is  so  simple  and  so  well  adapted  to  its  purpose 
that  it  has  quite  driven  the  others  out  at  the  present  time. 
It  consists  of  a  hollow  cylinder  exactly  turned  without  and 
blackened  within.  The  upper  end  is  drawn  suddenly  in  to  form 
a  raised  ledge  of  somewhat  less  diameter  than  the  opening  in 


86  THE   MICROSCOPE  IN  BOTANY. 

the  stage.  Over  this  upper  edge  of  the  cylinder  are  placed 
small  metal  caps  which  are  provided  with  central  openings  of 
various  sizes. 

[This  excellent  piece  of  apparatus,  which  has  not  yet  come 
into  very  general  use  in  this  country,  is  a  survival  of  the  "dark 
well"  or  "dark  chamber"  of  the  early  days  of  the  modern 
microscope.  It  may  be  slipped  from  below  into  a  ring  or  short 
tube  like  that  projecting  from  the  lower  surface  of  the  stage  in 
Plate  V,  or  supported  by  the  sub-stage,  as  in  Plate  X,  or  in 
microscopes  of  very  simple  construction  inserted  into  a  special 
carrier  to  be  presently  described.] 

3.  [  The  Iris  Diaphragm  is,  by  far,  the  most  perfect  means  of 
limiting  the  cone  of  illuminating  rays,  and  is  now  produced  in 
various  forms  by  numerous  makers.     That  of  the  Bausch  and 
Lomb  Optical  Co.  is  represented  in  Fig.  27.     It  consists  of  a 

dark  well,  closed  at  the  top  by  a  series  of  thin 
movable  plates,  which  by  a  very  simple  mechan- 
ism may  be  made  to  close  the  opening  altogether, 
or  to  open  gradually  and  form  a  practically 
circular  aperture  of  any  desired  size  up  to  the 
maximum  capacity  of  the  well,  the  size  of  the 
opening  being  controlled  by  the  milled  head  at 
FIG.  27.  the  bottom.  £uch  ease  and  precision  are  secured 

by  this  contrivance,  in  adjusting  the  amount  of  light  admitted, 
while  the  object  is  under  observation  and  all  the  adjustments  of 
the  stand  and  light  remain  undisturbed,  that  a  person  once 
accustomed  to  its  use  is  little  likely  to  be  satisfied  without  it. 
How  it  may  be  combined  with  tlje  condensing  lens  will  appear 
further  on.  R.  H.  W.] 

4.  [Special  Diaphragm-stops.']     All  the  contrivances  thus 
far  considered  are  adapted  to  shut  off  the  marginal  rays  which 
the    mirror  reflects    upon  the  object.     Latterly,  attention  has 
been  given  to  correct  the  central  rays  which  proceed  from  the 
mirror.     It  has  been  found  that  they  frequently  injure  the  mi- 
croscopic image ;  particularly  so  is  this  true   in  the  use   of  a 
condenser,  where  one  must  often  provide  for  their  elimination. 
This  can  be  done  very  simply  by  the  use  of  a  "central-stop." 
A  central-stop  is  a  small  circular  metal  plate  which  is  supported 


CONDENSEKS.  87 

on  the  slenderest  possible  metallic  arm  [or  on  a  thin  glass  disk], 
and  which  by  some  contrivance  may  be  brought  into  the  middle 
of  the  condensing  lens. 

[A  horizontal  slit,  consisting  of  a  central  opening  much 
longer  than  wide,  in  the  diaphragm  plate,  is  used  to  great  ad- 
vantage in  connection  with  the  condensing-lens,  in  the  illumi- 
nation of  binocular  microscopes,  sufficient  angular  breadth  of 
illumination  being  thus  secured  to  light  both  fields  of  the  instru- 
ment freely  without  such  an  excess  of  light  as  would  impair  the 
view  of  the  object.  A  corresponding  effect  is  attained  by  the 
use  of  a  pair  of  horizontally  arranged  circular  apertures  sepa- 
rated at  such  an  angular  distance  apart  that  each  one  will  admit 
the  pencil  of  light  required  by 
one  tube  of  the  instrument.* 
The  pair  of  apertures  requires 
to  be  adjusted  with  more  skill 
than  the  horizontal  slit,  and  ap- 
parently without  compensating 

FIG.  28. 
advantages.    With  these  stops, 

shown  in  Fig.  28,  the  use  of  the  binocular  may  be  extended  to 
higher  powers  and  angles  than  without  them.  Special  stops  for 
oblique  light  or  other  effects  may  be  used  at  the  option  of  the 
student.  Any  of  the  special  stops  may  be  cut  in  certain  portions 
of  the  revolving  diaphragm-plate  or  in  some  of  the  caps  of  the 
cylindrical  diaphragm  which  must  be  substituted  for  the  iris 
diaphragm  during  the  time  of  their  use.  R.  H.  TT.] 


C.     CONDENSERS. 

For  certain  purposes  it  is  recommended  to  interpose  a  lens 
between  the  mirror  and  the  object  which  will  concentrate  the 
rays  of  light  from  the  mirror  exactly  on  one  point.  This  can 
be  done  best  with  a  condensing  lens  of  very  short  focus.  We 
commonly  use  a  plano-convex  with  the  convex  side  strongly 
curved. 

[Such  a  condensing  lens  is  often  required,  not   only  to  in- 

*See  a  figure  and  description  of  this  method  of  binocular  illumination  by  the  writer  in 
the  American  Naturalist  of  December,  1870,  p.  636. 


88  THE   MICROSCOPE   IN  BOTANY. 

crease  the  amount  of  light  reaching  the  object,  but  also  to 
secure  effects  dependent  upon  the  obliquity  with  which  the  light 
passes  through  it.  The  simplest  arrangement  and  one  of  the  best 
is  a  nearly  hemispherical  lens,  10  to  12  mm.  in  diameter,  stuck 
to  the  bottom  of  the  object-slide,  directly  beneath  the  object, 
by  a  minute  quantity  of  glycerine  or  oil  of  cloves.  The  lens 
should  be  less  than  a  hemisphere  by  about  the  average  thick- 
ness of  an  object-slide,  so  that  when  the  two  are  united  the 
object  will  be  at  the  center  of  curvature.  Light,  either  par- 
allel from  the  plane  mirror  or  condensed  by  the  concave  mirror, 
may  then  be  passed  with  a  peculiarly  brilliant  effect,  directly  to 
the  object  from  the  mirror  in  whatever  position,  from  axial  to 
the  level  of  the  bottom  of  the  stage,  in  which  the  mirror  may 
be  placed.  If  the  obliquity  chosen  be  in  excess  of  the  semi- 
aperture  of  the  objective*  light  will  pass  through  the  object  but 
not  directly  into  the  objective  ;  the  object,  if  neither  too  opaque 
nor  too  translucent,  then  appearing  brilliantly  illuminated  upon  a 
dark  field,  the  same  effect  being  produced  with  less  intensity  by 
the  prism  mentioned  below,  or  with  very  limited  brightness  and 
only  for  very  low  powers,  by  the  concave  mirror  alone  in  a  very 
oblique  position.  The  condensing  power  of  the  hemisphere  is 
small,  on  account  of  its  large  curvature  and  the  position  of  the 
object  far  within  its  focus.  If  greater  refraction  be  desired,  the 
"  Wenham  button"  may  be  substituted,  whose  sharp  curvature 
and  more  precise  focus  give  a  more  intense  illumination,  but 
one  adequate  only  for  minute  objects  and  applicable  chiefly  to 
the  higher  powers.  For  illumination  with  parallel  instead  of 
converging  rays  a  small  triangular  prism  may  be  similarly  at- 
tached to  the  slide,  the  effects  being  the  same  except  that  the 
light  is  not  condensed  and  that  its  obliquity  is  limited  to  one 
angle,  or  if  the  prism  be  revolving  and  not  equilateral,  to  two 
or  three  angles.  Such  a  prism  upon  a  convenient  mounting  is 
shown  in  Fig.  29.  It  is  of  far  less  general  applicability  than 
the  hemispherical  lens.  Both  lens  and  prism  are  somewhat 
difficult  to  locate  exactly  in  the  required  position,  and  are  liable 
to  slip  out  of  place  if  the  stand  be  inclined,  especially  if  too 
much  of  the  connecting  liquid  be  employed.  For  these  rea- 
sons they  should  be  mounted,  for  stands  having  a  sub-stage,  at 


CONDENSERS.  89 

the  summit  of  a  vertical  wire  rising  from  the  center  of  the  sub- 
stage.  When  there  is  no  sub-stage,  this  wire  may  be  supported 
by  an  arm  attached  to  the  stage  itself,  as  in  the  ingenious 
device  of  Jas.  W.  Queen  &  Co.,  which  appears,  with  prism  at- 
tached, in  Fig.  29.] 

[If  the  lens  be  mounted  at  the  top 
of  a  dark  well,  its  angular  capacity 
will  of  course  be  limited  by  the  posi- 
tion of  the  lower  edge  of  the  tube 
which  should,  for  this  reason,  be  short 
and  broad.  It  can  then,  however,  be 
slipped  away  from  the  object,  down-  FIG  29 

wards,    its   glycerine    contact    being 

omitted,  and  focussed  upon  the  object  from  below.  In  this  case 
its  capacity  as  a  condenser  is  greatly  increased  by  placing  be- 
neath it  a  still  larger  lens  called  a  collecting  lens.  Condensers  of 
two  large  non-achromatic  lenses  have  been  extensively  used  with 
great  success  for  many  years,  largely  through  the  influence  of 
Dr.  Beale  in  advocating  the  use  of  an  ocular  for  that  purpose; 
and,  notwithstanding  recent  improvements  in  this  direction, 
one  may  still  with  much  satisfaction  transfer  his  highest  power 
ocular  to  a  ring  beneath  the  stage,  as  a  condenser.  In  the  or- 
tlioscopic  ocular,  similarly  used,  and  the  "  Webster"  condenser, 
an  achromatic  upper  lens  is  employed ;  while  in  the  latest  and 
now  most  approved  form,  introduced  by  Professor  Abbe  and 
hence  called  the  Abbe  condenser,  both  of  the  lenses  are  non- 
achromatic  and  of  such  great  thickness  that  the  top  of  the  lens 
will  nearly  touch  the  object-slide  when  focussed  upon  the  object. 
By  this  arrangement  not  only  is  great  aperture  (n.  a.  1.42  and 
upwards)  readily  obtained  for  use  with  the  highest-angled  ob- 
jectives, but  water  or  glycerine  contact  with  the  object-slide 
becomes  practicable,  giving  an  "immersion"  illuminator  with 
increased  working  capacity.  This  simple  and  inexpensive 
combination  seems  to  be  superseding,  with  good  reason,  all  the 
elaborate  and  carefully  corrected  achromatic  condensers  for- 
merly used.] 

[By  combining  a  black  center-stop  with  a  condensing  lens  or 
system,  a  central  cone  of  light  below  the   object  and  a  corre- 


90  THE   MICROSCOPE   IN  BOTANY. 

spending  inverted  cone  of  light  above  the  object  will  be  sup- 
pressed, each  having  the  same  angular  aperture  as  the  obstructed 
portion  of  the  condenser.  If  an  objective  of  less  than  this 
aperture  be  used,  it  will  of  course  receive  no  direct  light,  but 
will  view  the  object  illuminated  by  the  oblique  rays  from  the 
unobstructed  marginal  position  of  the  condenser,  the  field  mean- 
while remaining  dark.  For  low  powers  exclusively,  a  single 
thick  lens  with  a  central  black  stop  attached  to  its  upper  sur- 
face, known  as  the  spot  lens,  is  sufficient.  An  achromatic  con- 
denser with  center-stops,  or  the  Wenham  paraboloid,  a  truncated 
glass  paraboloid,  with  a  center-stop,  to  give  by  internal  reflec- 
tion a  hollow  cone  of  rays  condensed  at  a  large  angle,  has  been 
heretofore  employed  with  the  higher  powers ;  but  the  large 
lens-systems  of  the  different  varieties  of  Abbe  condensers, 
with  their  large  apertures  and  immense  amount  of  light,  if  pro- 
vided with  suitable  center-stops,  leave  little  to  be  desired,  with 
either  high  powers  or  low.  Objectives  of  too  large  aperture 
for  this  method  of  illumination  are  frequently  brought  within 
its  scope  by  inserting  a  diaphragm  behind  them  to  temporarily 
reduce  their  aperture  to  a  practicable  limit.  This  so-called 
black  ground  or  dark  field  illumination  is  very  effective  with 
many  delicate  vegetable  hairs,  fibres,  etc.,  which  should  usually 
be  viewed  dry  or  in  water,  as  balsam  renders  them  too  trans- 
parent to  arrest  and  disperse  sufficient  light.  R.  H.  W.] 

[  D.    ILLUMINATING  COMBINATIONS.  ] 

[1.  The  Universal  Accessory.  For  the  sake  of  convenience, 
some  of  the  opticians  combine  into  one  piece  of  apparatus,  to 
be  fitted  below  the  stage,  several  of  the  sub-stage  appliances, 
such  as  diaphragms,  condensing  lenses,  polarizing  prism,  etc. 
A  simple  arrangement  for  this  purpose,  being  inexpensive  and 
applicable  to  smaller  stands,  is  the  "Universal  Accessory"  of 
Bausch  and  Lomb,  shown  in  Fig.  30.  It  consists  of  a  rather  thick 
stage-plate  intended  to  lie  upon  the  stage  in  place  of  the  object 
slide,  and  to  carry  the  slide  under  a  pair  of  spring  clips  upon 
its  upper  surface.  Set  into  the  centre  of  this  plate  and  projecting 
below  it  through  the  central  opening  of  the  stage,  is  a  short 


ILLUMINATING  COMBINATIONS. 


91 


revolving  tube  to  receive  a  cylindrical  diaphragm,  polarizing 
prism,  or  condensing  lens ;  the  latter  becoming,  with  a  black 
centre  stop,  an  efficient  spot  lens.  This  apparatus  is  especially 
suited  to  stands  having  no  sub-stage  conveniences.] 


FIG. 


[2.  Ward's  Iris  Illuminator.  A  more  elaborate  and  effective 
arrangement,  constituting  an  illuminator  suitable  for  work  of  a 
higher  grade,  is  a  combination  of  the  condensing  lens  with  a 
decentering  iris  diaphragm,  devised  by  the  writer  and  made  by 
Bausch  andLomb.  It  is  shown  in  Fig.  31,  and  consists  of  any 


FIG.  31. 


or  "immersion,"  under  and 


desired  lens  system,  either  "  dry 
close  to  which  is  mounted  an  iris  diaphragm  set  in  a  sliding 
plate  so  that  it  can  be  moved  into  any  position  from  the  center 
to  the  periphery  of  the  system,  without  altering  the  position  of 
the  latter.  Thus  not  only  the  obliquity  of  the  light,  but  the 


92  THE   MICROSCOPE   IN   BOTANY. 

exact  amount  desired  or  found  advantageous  at  any  chosen  ob- 
liquity can  be  regulated  with  perfect  precision  by  a  touch  of  the 
hand  to  the  decentering  screw  and  to  the  adjusting  collar  of 
the  diaphragm.  This  contrivance  can  be  applied,  without  out- 
growing the  limits  of  the  customary  1J  inch  (38  mm.)  sub-stage 
tube  (which  size  of  sub-stage  is  being  very  generally  adopted, 
and  it  is  hoped  will  soon  be  made  "  standard") ,  to  any  condensing 
system  whose  posterior  diameter  does  notexceed  21  mm.  (|j  in.) . 
It  is  well  adapted  to  a  simple  hemispherical  lens,  a  large-lens 
±  achromatic  condenser,  or  the  doublet  of  thick  non-achromatic 
lenses  adopted  by  Prof.  E.  Abbe  of  Jena.  In  using  the  first 
or  the  last  of  these  three,  which  have  nearly  superseded  the  late 
"achromatic  condensers,"  it  should  not  be  forgotten  that  the 
best  performance  is  nearly  always  obtained  by  connecting  the 
illuminating  lens  with  the  object-slide  by  a  drop  of  water. 
A  blue  glass  disk,  for  correcting  the  glare  and  color  of  artificial 
light,  is  fitted  to  a  tube  that  can  be  inserted  into  the  bottom  of 
the  dark  well  of  the  diaphragm.  A  special  adapter  is  also  pro- 
vided for  the  use,  in  place  of  the  iris  diaphragm,  of  central  stops 
for  securing  dark  field  illumination  ;  and  a  revolving  tube,  slipping 
inside  of  this,  carries  a  horizontal  slit,  or  pair  of  horizontally 
arranged  apertures,  for  the  better  illumination  of  binocular  mi- 
croscopes (see  page  40),  or  special  stops  for  the  production  of 
any  effect  desired  by  the  user.  In  similar  fittings,  may  be 
mounted  a  polarizing  prism  and  selenite  plate,  a  small  Nicol's 
prism  being  sufficient,  in  connection  with  the  condenser,  to  give 
adequate  illumination  for  moderately  high  powers.  The  whole 
apparatus  rotates  about  its  own  optical  axis,  which  remains 
coincident  with  that  of  the  microscope  itself.  By  removing  the 
lenses  from  the  top  of  the  apparatus,  the  iris  diaphragm,  with 
or  without  its  blue  glass  disk,  or  the  polarizer,  will  be  found 
in  position  for  use  by  itself.  Except  for  very  low  powers, 
however,  the  illuminator  may  be  considered  as  a  part  of  the 
stand  and  kept  habitually  in  place,  the  changes  of  light  required 
for  a  great  variety  of  work  being  readily  accomplished  by  its 
aid.  It  can  be  applied  to  almost  any  microscope,  whether  with 
or  without  a  sub-stage.] 


OPAQUE  ILLUMINATORS. 


93 


E.    OPAQUE  ILLUMINATORS. 

[The  foregoing  methods  of  illumination  pertain  to  objects 
sufficiently  transparent  or  translucent  to  be  viewed  by  light 
passed  through  them  from  below.  Opaque  objects,  viewed  by 
light  reflected  from  their  upper  surface,  as  frequently  becomes 
necessary  in  botanical  study,  can  seldom  be  adequately  illumi- 
nated by  the  general  light  of  the  room.  They  usually  require 
for  satisfactory  exhibition  the  condensation  of  light  upon  them 
by  means  of  a  lens  or  mirror.  A  small  condensing  lens  may  be 


FIG.  32  a 


FIG.  32  b 


attached  to  the  microscope  itself,  or  mounted  upon  a  stand  of 
its  own.  Large  plano-convex  condensing  lenses  called  "bull's 
eyes,"  having  great  thickness  and  short  focal  distance,  are 
usually  mounted  on  a  separate  stand  as  shown  in  Fig.  32.  They 


94  THE  MICROSCOPE  IN  BOTANY. 

are  used  near  the  microscope,  the  object  being  in  the  principal 
focus,  to  condense  light  upon  the  object,  or  near  a  lamp,  the 
flame  being  in  the  principal  focus,  to  give  a  beam  of  intense  and 
nearly  parallel  rays  for  use  at  a  convenient  distance  from  the 
flame.  In  stands  having  the  modern  style  of  swinging  tail- piece, 
as  in  Plates  III  to  XI,  the  concave  mirror 
can  be  readily  brought  above  the  stage  for 
the  concentration  of  light  upon  the  object. 
For  higher  powers  a. small  concave  silvered 
mirror,  or  side  reflector,  is  attached  to  the 
objective  or  to  some  neighboring  part  of  the 
stand.  Opaque  objects  under  the  highest 
powers  can  be  sufficiently  lighted  by  mak- 
ing the  objective  its  own  condenser,  as 
in  the  case  of  the  Beck  illuminator,  shown  in  Fig.  33,  where 
light  entering  an  aperture  in  the  side  of  the  nose-piece  is  reflected 
downward  by  a  thin  cover-glass  in  the  center  of  the  tube,  and 
is  condensed  by  the  objective  upon  the  object  in  its  focus.  In 
the  use  of  such  contrivances,  of  which  there  are  many  in  use, 
much  care  and  tact  are  necessary  to  avoid  the  glare  of  false  light. 
R.  H.  W.] 

F.     OBSERVATION  BY  ARTIFICIAL  ILLUMINATION. 

The  microscope  is  an  apparatus  of  the  day.  The  best  source 
of  light  for  it  is  diffused  daylight,  when  the  heavens  are  evenly 
covered  with  a  transparent  white  veil  of  clouds.  The  blue  of 
the  cloudless  heavens,  the  changing  light  of  hurrying  clouds, 
and  the  direct  sunlight  are  alike  objectionable  for  careful  micro- 
scopical investigations.  But  if  one  has  necessary  microscopical 
work  to  do  on  a  sunny  cloudless  day,  his  best  course  is  to  take 
a  white  wall  or  a  large  white  paper  screen  which  reflects  sunlight 
as  the  source  of  his  illumination. 

But  it  will  now  and  then  happen  to  the  microscopist  that  he 
must  work  by  the  light  of  the  lamp,  it  may  be  to  make  the 
evening  hours  help  out  the  short  dark  winter  days,  or  it  may  be 
to  study  the  reproductive  processes  in  the  lower  cryptogams, 
which  take  place  only  during  the  night  hours.  In  these  cases  one 


OBSERVATION  BY  ARTIFICIAL  ILLUMINATION.  95 

has  to  contrive  some  way  to  improve  the  artificial  light  which  in 
and  of  itself  is  in  the  very  highest  degree  unserviceable  for  mi- 
croscopical observations.  The  yellow  light  of  gas  or  petroleum 
consists,  for  the  most  part,  of  rays  which  belong  to  the  first 
half  of  the  spectrum  and  are,  more  than  any  other,  hurtful  to 
the  eyes  of  the  observer.  Besides,  if  one  turns  the  mirror  di- 
rectly to  the  flame,  the  field  of  view  is  commonly  too  bright, 
and  if  toward  the  lamp  shade,  it  is  too  dark.  In  order  conven- 
iently to  modify  the  brightness  and  color  of  the  light,  the 
following  means  is  suggested  and  recommended.  Direct  the 
mirror  to  the  brightest  part  of  the  flame  and  then  interpose, 
close  to  the  mirror,  an  upright  screen  of  thin  translu- 
cent paper,  which  one  can  easily  make  in  a  few  moments. 
This  subdues  the  brightness  of  the  field  so  much  as  to  make  it 
painless  to  the  eyes.  In  order  to  change  the  yellow  color  to  a 
blue  tint  it  is  only  necessary  to  bring  between  the  mirror  and 
the  diaphragm,  a  little  plate  of  blue  cobalt  glass.  I  use  a  little 
round  glass  disk,  about  13  mm.  in  diameter  and  2  mm.  thick.  Its 
color  is  a  faint  blue  and  the  underside  is  slightly  and  uniformly 
ground.  This  little  plate  is  placed  directly  under  the  diaphragm. 
In  this  way  the  illumination  of  the  field  is  made  uniform  and 
•  soft  bluish. 

The  following  arrangement  is  still  better  and  more  convenient. 
Fill  an  ordinary  cobbler's  globe*  with  a  pretty  dark  blue  solution 
of  cupric  oxide  of  ammonia,  and  place  it  between  the  source  of 
light  and  the  mirror  in  such  a  way  that  the  mirror  can  be  directed 
towaid  the  brightest  spot  of  white  light  produced.  In  this  way 
one  gets  an  illumination  for  evening  which  leaves  very  little 
to  be  desired.  The  right  concentration  of  the  cupric  oxide  of 
ammonia  is  easily  made  out  after  a  few  trials. 

XIII.     THE  MICROSCOPE  FOOT. 

Of  the  microscope  foot  two  things  are  required.  The  mi- 
croscope must  rest  on  it  firmly  and  securely.  The  foot  should 

*I  am  told  that,  in  Germany,  the  cobblers,  when  at  work  in  the  evening,  suspend  a 
glass  globe  filled  with  water  between  themselves  and  the  lamp,  which  seta  upon  the  table 
before  them,  in  such  a  position  that  it  concentrates  the  light  directly  upon  their  work. 
A.  B.  H. 


96  THE  MICROSCOPE  IN  BOTANY. 

be  neither  too  light  nor  too  small.  Formerly  the  microscope 
foot  was  made  in  a  disk  or  plate-like  form,  of  a  thin  plate  of  brass 
loaded  with  lead.  But  to  this  form  of  foot  there  was  this  objec- 
tion, that,  unless  the  surface  upon  which  it  rested  was  exactly 
flat  and  even,  the  microscope  would  totter  or  wabble  with  every 
motion.  So  at  the  present  time  the  microscope  is  made  with 
the  horse-shoe  foot  [or  with  a  tripod  giving  three  widely  sepa- 
rated points  of  support.  The  latter  effect  is  most  frequently 
gained  by  means  of  three  solid  projections  on  the  bottom  of  the 
brass  plates  or  arms  as  shown  in  all  the  Plates  but  especially 
in  Nos.  V,  X  and  XL  It  is  now  customary  to  give  more 
satisfactory  means  of  contact  with  the  table  by  supplying  the 
feet  with  disks  of  soft  India  rubber  projecting  slightly  from  their 
lower  surface.  R.  H.  W.] 

The  folding  microscope  foot,  which  consists  of  three  legs,  is 
altogether  objectionable.  '  They  never  give  a  firm  support  and 
are  the  most  impracticable  things  ever  devised  for  the  microscope. 
At  best  this  form  is  applicable  only  to  travelling  microscopes 
where  its  compendiousness  is  its  only  recommendation. 


XIY.  RULES  FOR  THE  USE  OF  THE  MICROSCOPE. 

A  good  instrument  is  a  very  precious  article,  but  a  microscope 
ruined  through  neglect  is  the  most  hateful  thing  one  can  see. 
By  proper  treatment  a  microscope,  though  much  and  often  used, 
will  remain  unchanged  for  many  long  years,  assuming  that  it 
be  not  too  much  exposed  to  its  mortal  foe,  the  dust,  and  that 
it  be  cleaned  each  time  after  using.  It  may  not  be  superfluous 
here  to  add  some  hints  about  keeping  the  microscope  clean. 

It  is  an  easy  matter  to  keep  the  metallic  part  clean.  It  is 
only  necessary  after  each  using  to  rub  it  carefully  with  chamois 
skin  or  linen.  Commonly  it  will  be  sufficient  to  rub  it  dry,  to 
restore  the  brightness,  but  sometimes  water  may  be  used  espe- 
cially in  cleaning  the  stage.  Alcohol  and  the  like  should  under 
no  circumstances  be  used  on  the  polished  parts  of  the  micro- 
scope, because  it  will  be  sure  to  dissolve  the  lacquer  with  which 
they  are  covered.  But  its  use  is  not  necessary,  since  no  oil  or 


RULES  FOR  THE  USE  OF  THE  MICROSCOPE.  97 

glvcerine  or  the  like  should  bs  put  on  the  screws  or  any  part  of 
the  stand.  The  dull  parts  of  the  oculars  so  far  as  they  sink 
into  the  microscope  tube,  the  tube  as  far  as  it  runs  in  the  outer 
sheath,  and  finally  the  diaphragm  cylinder,  should  be  carefully 
cleaned. 

Keeping  the  glasses  cleaned  demands  still  greater  care. 
Many  people  believe  the  optical  parts  of  the  microscope  are 
clean  when  no  dust  can  be  seen  in  the  field  of  vision.  But  this 
is  proof  only  that  the  under  ocular  glass,  the  collecting  or  field, 
lens  is  clean.  Particles  of  dust  on  the  objective  cannot  be  seen: 
in  the  field  of  vision  as  a  little  reflection  will  show,  but  they 
exercise  a  damaging  influence  on  the  microscopic  image,  since 
they  cause  a  cloudiness  and  impaired  definition.  It  is  therefore 
necessary  to  clean,  occasionally,  both  oculars  and  objectives. 
For  this  purpose,  old,  very  soft  linen  washed  repeatedly  in  dis- 
tilled water  or  a  soft  hair  pencil  with  distilled  water  should  be 
used. 

The  ocular  should  be  cleaned  in  the  following  way.  Suppos- 
ing it  to  be  quite  soiled,  both  glasses  should  be  unscrewed 
and  first  of  .all  wiped  with  dry  linen.  Then  dampen  a  clean 
piece  of  linen  with  distilled  water  and  rub  each  glass  holding  it 
by  both  edges.  This  may  be  repeated  as  often  as  necessary, 
and  when  it  is  rubbed  perfectly  dry  it  should  be  swept  with  a 
fine  hair  pencil  to  remove  any  fibres  from  the  linen  which  may 
be  adhering  to  it.  In  this  way  we  get  perfectly  clean  glasses. 
To  test  the  purity  of  a  cleaned  glass  it  should  be  breathed  upon 
a  little.  If  it  is  perfectly  clean  the  dampness  will  all  disappear 
at  the  same  time.  But  if  there  is  a  particle  of  dust  it  will 
gather  in  a  little  zone  about  that  and  evaporate  there  a  little 
later  than  it  does  on  the  clean  surface. 

The  objective  is  cleaned  in  the  same  manner,  but  since  its 
three  lenses  are  joined  in  an  air-tight  mounting  it  is  scarcely 
possible  for  dust  to  collect  between  them.  So  we  shall  have  to 
clean  only  the  upper  and  under  surface  of  the  lens-system. 
Usually  it  may  be  done  as  with  the  oculars.  But  since  the 
upper  object  glass  is  difficult  to  come  at  we  should  prepare  «i 
wooden  stick  with  some  soft  linen  bound  over  the  end  of  it, 
and  with  this  reach  down  and  clean  off  the  glass.  Should  the 

7 


98  THE  MICROSCOPE  IN  BOTANY. 

lower  lens  get  besmeared  by  the  careless  use  of  the  reagents  it 
should  be  at  once  cleaned  by  repeated  washings  with  distilled 
water.  Water  will  suffice  in  most  cases.  But  if  it  be  necessary 
to  use  alcohol  it  should  be  done  with  the  greatest  caution  and 
celerity.  For  by  employing  alcohol  we  are  in  the  greatest 
.danger  of  ruining  the  whole  system,  by  the  alcohol  penetrating 
within  the  mounting  of  the  glasses  and  partly  dissolving  the 
Canada  balsam  by  which  the  crown  and  flint  glass  lenses  are 
cemented  together.  The  cleanliness  of  the  objective  may  be 
tested  in  the  same  way  as  the  ocular,  as  indicated  above,  or  by 
examining  the  reflected  image  of  a  window  in  the  glass  by 
means  of  a  magnifying  lens. 

For  the  preservation  of  the  microscope,  besides  cleanliness, 
the  handling  of  the  screws  is  of  importance.  This  particularly 
concerns  the  matrix  of  the  tube  and  the  screw  of  the  objective- 
system  which  fits  into  it.  Since  by  almost  every  change  in  the 
magnification  this  must  be  screwed  on  and  off,  and  since  by 
putting  the  objective  on  a  little  obliquely,  the  whole  screw 
arrangement  may  easily  be  injured,  the  following  method  of 
screwing  on  the  pails  is  commended  as  one  certain  to  work  no 
injury  to  the  apparatus.  The  objective  should  be  set  upon 
the  tube  with  its  thread  close  up  against  the  end ;  then  it  should 
be  turned  backwards,  as  in  the  act  of  unscrewing,  till  one  hears 
the  short  click  which  shows  that  the  thread  has  fallen  into  its 
proper  place,  when  the  system  may  be  reversed  and  screwed 
up. 

Further,  the  greatest  care  should  be  used  in  focussing.  With 
high  magnifications  the  objective  is  brought  very  close  to  the 
object  and  must  be  carefully  guarded  in  the  act  of  focussing,  or 
both  objective  and  object  will  be  ruined.  Many  microscopists 
do  the  focussing  in  this  way.  The  tube  is  pushed  down  by 
hand,  or  by  the  rack  and  pinion,  till  it  comes  very  near  to  the 
objects  and  then  exactly  focussed  with  the  fine-adjustment  screw. 
With  the  use  of  low  powers  this  is  satisfactory,  but  with  higher 
magnifications,  and  in  the  use  of  the  lower  by  beginners,  the 
following,  reverse,  method  is  recommended.  By  looking  across 
the  stage  from  the  side  one  can  bring  the  objective  down  very 
close  upon  the  preparation,  still  without  touching  it,  bring  it 


RULES  FOR  THE  USE  OF  THE  MICROSCOPE.  99 

within  the  focal  distance.  Then  by  hand  or  by  the  rack  and 
pinion  run  the  tube  upward  till  the  coarse  adjustment  is  reached 
and  finish  with  the  fine-adjustment  screw.  This  method  secures 
perfect  safety  for  both  lens  and  specimen. 

Respecting  the  preparation  to  be  examined,  it  is  clear  that 
both  slide  and  cover-glass  should  be  perfectly  clean  before 
it  is  put  under  the  lens.  Permanent  preparations  should  be 
wiped  each  time  before  using,  with  a  piece  of  linen,  and  if 
they  are  used  with  an  immersion  fluid  it  should  be  carefully 
removed  after  each  using. 

If  a  preparation  is  to  be  studied  for  a  considerable  time  with- 
out removal  from  the  stage,  a  glass  bell  should  be  placed 
over  the  microscope  and  preparation,  which,  in  case  it  rests 
upon  the  same  leather  or  cloth-covered  wooden  plate,  with  the 
microscope,  will  effectually  exclude  the  dust. 

If  one  brings  the  microscope  in  winter  from  a  cold  to  a  warm 
room  and  undertakes  to  use  it  at  once,  the  ocular  glass  becomes 
dimmed  by  the  condensation  upon  it  of  vapor  from  the  body  or 
the  atmosphere.  It  is  better,  therefore,  to  bring  in  the  mi- 
croscope some  little  time  before  the  work  is  to  begin,  and  set  it 
near  the  stove  to  warm  up. 

If  the  microscope  is  to  be  for  a  long  time  out  of  use,  it  should 
be  inclosed  in  its  mahogany  case,  and  put  away  in  some  closely 
shutting  cupboard  in  which  is  placed  a  little  dish  of  chlorate  of 
lime.  This  insures  the  safety  of  the  steel  parts  from  rust,  and 
prevents  the  formation  of  verdigris.  Never  should  the  micro- 
scope, and  under  no  circumstances  should  the  objectives,  be 
stored  in  a  closet  in  which  the  reagents  ar^  kept,  for  out  of  the 
closest-stoppered  reagent  bottle,  there  will  come  some  vapor  of 
acid,  which  will  at  length  cause  the  greatest  injury  to  every 
kind  of  optical  apparatus. 


CHAPTER  II. 
MICROSCOPICAL  ACCESSORIES. 


UNDER  this  term  we  include  a  series  of  implements  which 
finds  frequent  use  in  microscopical  investigations.  The  greater 
part  of  them  are  connected  with  the  optical  apparatus  of  the 
microscope  itself.  The  most  important  microscopical  acces- 
sories of  which  we  shall  here  speak  are  the  preparing  micro- 
scope, the  apparatus  for  drawing  or  photographing  microscopic 
pictures,  the  micrometer,  the  polarizing  apparatus,  the  goni- 
ometer and  the  micro-spectroscope. 


I.  THE  PREPARING  MICROSCOPE. 

(DISSECTING  OR  MOUNTING  MICROSCOPE.) 

The  preparing  microscope  is  of  use,  in  order  to  prepare  those 
objects  which  have  previously  been  made  ready  for  examination 
by  means  of  the  section-cutting  instrument,  and  need  to  be 
examined  first  with  a  low  power,  in  order  to  lay  them  rightly  on 
the  object-slide,  or,  by  means  of  small  needles  and  knives,  to 
further  treat  and  manipulate. 

1.  The  Simplex.  The  preparing  microscope  consists  essen- 
tially of  a  mounted  magnifying  glass  in  the  focus  of  which  the 
slide  bearing  the  object  may  be  brought ;  beneath  this  is  placed 
the  mirror  to  furnish  the  necessary  light  for  the  object. 

A  simple  magnifying  glass  gives  comparatively  but  small 
magnification.  To  get  strong  magnifying  power  it  would  be 
necessary  to  make  the  lens  with  a  high  superficial  curvature, 
and  this  would  involve  a  short  focal  distance  and  too  great  prox- 
imity to  the  object  to  allow  a  convenient  use  of  the  dissecting 
needles.  For  this  reason  we  have  for  a  long  time  ceased  to  use 

(100) 


THE  PREPARING  MICRO  SG.OPE/ 


simple  magnifying  glasses  and  instead  use  a  combination  of  two 
or  three.  By  this  means  we  secure  a  shortening  of  the  focus 
and  greater  magnification  without  diminishing  the  working  dis- 
tance between  the  object  and  the  under  lens.  Combinations 
of  two  or  three  of  these  glasses  are  called  respectively  doublets 
and  triplets. 

[For  the  lowest  powers  of  the  preparing  microscope,  single 
lenses,  either  double-convex  or  plano-convex,  of  from  two  inch 
to  one-half  inch  (51  to  13  mm.)  focus  are  commonly  employed. 
By  some  of  the  makers  they  are  so  constructed  that  two  may  be 
used  together.] 

[2.   The  Coddington  Lens.     For  powers  as  high  as  one-fourth 
inch  (6  mm.)  and  as  low  even  as  one  inch  (25  mm.)  the  Cod- 
dington lens,  Fig.  34,  is   available. 
This  is  a  solid  glass  cylinder  whose 
ends  are  ground  to  spherical  surfaces 
both  of  which  are  portions  of  the  same 
sphere,  the  center  of  the  curvature  of 
both  being  identical  and  situated  in 
the  center  of  the  glass.      A  groove  FlG  34 

is  ground  around  the  circumference  of 

the  cylinder  and  blackened  to  serve  the  purposes  of  a  diaphragm. 
This  construction  gives  an  excellent  definition  which  is  "less 
dependent  than  in  any  other  magnifier  on  the  exact  adjustment 
of  the  lens  ;  since,  having  an  optical  axis  in  any  available  direc- 
tion, oblique  rays  pass  through  it  under  exactly  the  same  condi- 
tions as  axial  ones,  and  the  performance  is  therefore  not  marred 
by  imperfect  centering.  The  working  focus  is  rather  short. 
To  partially  remedy  this  fault  and  to  render  the  instrument  less 
clumsy,  the  cylinder  is  often  shortened,  at  a  slight  optical 
disadvantage,  by  bringing  the  convex  surfaces  nearer  together 
than  when  in  their  theoretical  position.] 

[3.  The  Achromatic  Triplet.  When  a  magnifier  having  the 
external  form  of  a  shortened  Coddington  is  made  achromatic,  it 
consists  of  two  double  convex  or  meniscus  lenses  of  large 
curvature,  whose  aberrations  are  corrected  by  a  thick  lens  of 
glass  of  a  different  refractive  index  cemented  between  them 
*  with  Canada  balsam.  The  "  Globe  lens  "  of  Gimdlach  is  literally 
an  achromatic  Coddington,  as  it  is  a  sphere  of  flint  glass, 


MICROSCOPE  IN  BOTANY. 


ground  hollow  and  filled  and  corrected  by  a  much  smaller 
sphere  of  crown  glass,  the  whole  being  reduced  to  a  cy- 
lindrical form  by  cutting  away  the  unused  peripheral  portion. 
Being  achromatic  it  does  not  require  the  diaphragm  groove. 
A  more  common  combination  is  a  pair  of  double  convex  crown 
glasses  corrected  by  a  thick,  double  concave  flint  glass ;  though 
some  makers,  notwithstanding  the  disadvantage  of  exposing 
the  softer  glass  to  outside  wear,  prefer  to  place  the  flint, 
in  the  form  of  a  meniscus,  at  each  end  of  the  crown.  This 
combination,  more  or  less  modified  and  variously  named  by 
different  makers,  constitutes  the  achromatic  triplet  which  is 
now  taking  the  place  of  all  other  magnifiers,  especially  for 
the  higher  powers,  in  the  simple  microscope.  It  gives  a  long 
working  focus  and  a  broad,  clearly  defined  and  beautifully 
lighted  field  of  view,  which  is  a  luxury  for  all  purposes  and 
may  be  considered  indispensable  for  very  fine  or  difficult 
work.  It  is  usually  mounted  like  the  Coddington,  Fig*  34. 
The  achromatic  objective  of  the  compound  microscope  is  not 
equally  suitable  for  use  in  the  simple  microscope,  though  the 
low  powers,  if  mounted  short,  are  sometimes  so  employed. 
For  this  purpose  the  separating  objectives  in  short  tubes,  Fig. 
2,  are  available.] 

[4.   The  Engraver's    Glass,  consisting  of  a   pair  of  plano- 
convex lenses,  about  45  mm.  in  diameter,  mounted  in  a  deep, 

hard  rubber  cell,  as  repre- 
sented in  Fig.  35  and 
forming  a  doublet  of  great 
size,  gives  a  large  field  of 
view,  is  used  with  little 
strain  or  fatigue  to  the  eye, 
and  is  a  very  serviceable 
preparing  microscope  when 
only  the  lowest  powers  are 
required.  The  writer  has 
employed  these  glasses  for 

twenty  years  with  great 
FIG.  35.  J  J 

satisfaction  in  the  exami- 
nation of  pressed  plants  mounted  upon  paper  as  herbarium 
specimens,  in  the  preliminary  examination  of  hand- writ  ing,  and 


THE  PREPARING  MICROSCOPE. 


103 


in  the  selection  from  among  large  masses  of  material,  as  of 
fabrics  or  mixed  fibers  or  other  substances  supposed  to  contain 
inequalities  or  adulterations,  the  portions  requiring  further  in- 
vestigation ;  also  in  performing  under  the  lenses  such  manipula- 
tions or  dissections  as  require  only  a  low  amplification.  Such 
lenses  may  be  best  supported  upon  the  large  lens  holder  shown 
in  Fig.  37.] 

[5.  The  Handy  Dissecting  Microscope.  A  preparing  mi- 
croscope of  extreme  simplicity,  made  by  Bausch  and  Lomb, 
is  shown  at  §  its  natural  size  in  Fig.  36.  It  consists  of  a  glass 
plate  into  which  is  screwed  an  upright  brass  rod  B,  which  sup- 
ports the  magnifiers  at  A.  These  are  three  simple  magnifying 
glasses,  capable  of  being  used  singly  or  together,  and  mounted 


FIG.  36. 


in  a  form  available  for  ordinary  pocket  use.  The  addition  of  a 
Coddington  lens,  Fig.  34,  suitably  mounted  to  be  attached  to 
the  same  stem,  gives  a  somewhat  higher  power,  of  good  quality. 
[6.  The  Lens  Holder.  Finding  the  lens  holders  in  use 
to  be  of  too  limited  applicability,  being  too  light,  for  instance, 
to  carry  the  large  engravers'  lenses,  and  too  short-armed  for 
the  convenient  study 'of  handwriting  upon  large  sheets  of  paper 
or  of  mounted  herbarium  specimens,  or  else  too  unstable  for  use 
with  higher  powers,  the  writer  has  devised  and  employed  a 


104 


THE  MICROSCOPE  IN  BOTANY. 


form,  .arranged  somewhat  like  the  stands  used  by  engravers, 
which  is  (unlike  them)  sufficiently  firm  and  manageable  for  either 
large  or  small  magnifiers,  of  low  or  high  powers,  and  is 
available  for  an  arm-length  of  20-25  cm.  It  consists,  as  shown 
in  Fig.  37,  of  a  rectangular  frame  which  slips  over  the  pillar 
of  a  bull's  eye  stand,  both  it  and  the  bull's  eye  being  often 
mounted  upon  the  same  stand,  for  the  sake  of  simplifying  the 
apparatus,  and  because  they  are  often  advantageously  used  in 
combination.  The  frame  slides  smoothly  up  and  down  the 
pillar,  being  held  in  any  position  by  an  included  spring.  To  an 


FIG.  37. 

extension  of  the  bottom  of  the  frame  is  attached  a  horizontal 
arm,  having  first  a  horizontal  pivot  joint,  and  secondly  a  ball 
and  socket  joint,  the  tension  of  these  being  readily  adjustable 
by  means  of  a  screw  with  a  large  milled  head.  By  bending  the 
joints,  the  lens  maybe  brought  near  the  pillar  for  use  in  connec- 
tion with  the  bull's  eye  ;  or  by  attaching  the  jaws  or  ring  to  a 
longer  wire,  the  total  arm  length  may  be  increased  at  will.] 


THE  PREPARING  MICROSCOPE. 


105 


[At  the  end  of  the  arm  rises  a  vertical  pivot  upon  which  can 
be  slipped  almost  any  kind  of  pocket  magnifier,  such  as  a  Cod- 
dington,  or  achromatic  triplet  Fig.  34,  or  a  three-lens  system 
like  A,  in  Fig.  36,  or  a  double  bellows-shaped  arrangement 
like  that  shown  in  situ  on  the  arm.  Or,  the  lenses  being 
removed,  a  split  wire  may  be  inserted  into  the  hollow  end  of 
the  arm,  bearing  a  pair  of  hinged  semicircular  jaws,  shown  in 
the  figure,  for  carrying  an  engraver's  glass,  or  any  variety  of 
large  lenses  not  requiring  delicate  adjustment.  For  magnifiers 
of  higher  power,  requiring  more  precise  adjustment,  a  ring  is 
substituted  for  the  jaws.] 


FIG. 


[There  is  a  fine  adjustment  at  the  top  of  the  rectangular 
frame,  where  a  screw  with  milled  head,  pressing  the  pillar 
against  the  spring,  promptly  but  steadily  depresses  the  lenses 
to  the  extent  of  about  four  times  its  own  motion.] 

[This  apparatus  is  now  made  for  the  trade  by  theBausch  and 
Lomb  Optical  Co.  When  supplied  with  fine  achromatic  lenses, 
and  kept  standing  always  ready  upon  the  table,  it  becomes  con- 


106 


THE  MICROSCOPE  IN  BOTANY. 


stantly  useful  even  to  persons  well  supplied  with  elaborate  ap- 
paratus. It  is  worked,  if  transmitted  light  be  required,  over 
the  stage  of  any  dissecting  microscope  that  may  be  within 
reach.  By  turning  the  jaws  or  ring  into  a  vertical  position, 
it  is  well  adapted  to  the  examination  of  living  aquatic  plants  in 
a  glass  jar  or  aquarium  ;  for  which  purpose  powers  of  50  to  100 
diameters  may  become  available  by  using  the  Briicke  magnifier 


FIG.  39. 


(p.  107)  or  the  Bausch  and  Lomb  compound  dissecting  magni- 
fier (p.  108),  which  for  this  use  should  be  screwed,  not  slipped, 
into  the  ring.] 

[7.  The  Compact  Dissecting  and  Mounting  Microscope  is 
made  by  Bausch  and  Lomb,  and  is  a  preparing  microscope  of 
medium  size  and  cost,  and  of  sufficiently  portable  form  for 
pocket  use.  It  is  represented  in  Fig.  38.  A  japanned  iron 
base,  9  cm.  square,  carries  a  mirror  in  a  central  location  for  axial 


PL.    XII. 


Compact  Compound  Dissecting  Microscope.     Bausch  &  Lomb. 


THE  PREPARING  MICROSCOPE.  107 

illumination,  and  also  supports  a  pillar  which  carries,  at  a  height 
of  9  cm.,  a  stage  of  blackened  brass,  slightly  smaller  than  the 
base.  This  stage  has  a  central  opening  of  about  32  mm.,  sup- 
plied with  a  removable  glass  plate.  Inside  the  pillar  is  a  trian- 
gular rack-bar  moved  by  a  pinion  with  a  milled  head,  which 
carries  a  transverse  bar  with  a  ring  at  the  farther  end,  into 
which,  in  the  optical  axis  of  the  instrument,  the  amplifying 
lenses  are  inserted.  This  ring  is  also  furnished  with  the  society 
screw,  by  means  of  which  objectives  may  be  substituted  for  the 
simple  lenses.  The  mirror  can  be  instantly  transferred  to  the 
bottom  of  the  stage  for  oblique,  or  to  the  top  of  the  stage  for 
opaque  illumination.  Hand  rests  after  the  German  style  can 
be  attached  to  the  sides  of  the  stage,  as  figured  in  Plate  XII. 
Both  base  and  stage  can  be  folded  flat  against  the  pillar,  in 
order  to  be  packed  in  a  very  small  case.] 

[8.  The  Botanical  Dissecting  Microscope,  Fig.  39,  made  by 
Mr.  Zentmayer,  is  a  somewhat  larger  and  heavier  instru- 
ment than  the  one  just  described  ;  but  it  has  essentially  the 
same  parts  similarly  arranged.  The  circular  base  is  12  cm.  in 
diameter,  and  the  stage  is  9  X  12  cm.,  with  a  central  opening 
4cm.  With  the  stage  increased  to  11  X  15cm.,  and  a  larger 
mirror  substituted,  compactness  would  be  still  further  sacri- 
ficed, but  the  Avorking  capacity  would  be,  in  the  writer's  opinion, 
correspondingly  increased.] 

[9.  The  Briicke  Compound  Dissecting  Microscope.  A  sepa- 
rating achromatic  objective,  whose  lenses  can  be  used  either 
sing-ly  or  together,  with  an  ocular  in  the  form  of  a  concave  eye 
lens  (as  originally  proposed  by  Prof.  Briicke  of  Vienna)  in- 
serted into  a  small  tube  sometimes  less  than  5  cm.  high,  which 
acts  as  a  little  compound  body,  constitutes  a  microscope,  upon 
the  principle  of  the  Galilean  telescope,  which  has  long  been 
used  in  France  and  Germany,  and  to  a  more  limited  extent  but 
not  Avith  less  satisfaction,  in  this  country.  The  objective  lenses 
commonly  used,  by  themselves  give  powers  of  from  12  to  30 
diameters,  which  poAvers  are  increased  by  the  eye  lens  to  from 
40  to  100  and  upwards.  With  the  highest  powers  the  working 
focus  is  exceedingly  long  (8  mm.),  affording  ample  room  for  the 
use  of  needles  or  dissecting  instruments.  The  field  of  view  is 
rather  small.] 


108  THE  MICROSCOPE  IN  BOTANY. 

[10.  The  Bausch  and  Lomb  Compound  Dissecting  Microscope. 
This  form,  just  introduced  as  a  substitute  for  the  Briicke  instru- 
ment, consists  of  a  little  compound  body  (combined  with  their 
compact  microscope  in  Plate  XII)  only  75  cm.  long  and  19  mm. 
in  diameter,  which  contains  a  diminutive  objective,  ocular  and 
erector.  By  the  use  of  the  draw-tube  the  erector  can  be  car- 
ried from  near  the  objective  to  near  the  top  of  the  body,  giving 
a  range  of  powers  of  from  12  to  150  diameters,  the  working  focus 
meanwhile  varying  inversely  from  38  to  6  mm.  The  light  trans- 
mitted is  less  than  with  the -Briicke  apparatus,  the  field  of  view 
averaging  about  the  same  for  the  same  powers.  The  advan- 
tage of  the  new  arrangement  is  the  ready  command  of  the 
whole  range  of  intermediate  powers  by  simply  sliding  the  draw- 
tube.  For  the  higher  powers,  it  may  be  used  without  the 
erector  if  preferred.  The  objective  is  a  dividing  one,  whose 
lenses,  the  compound  body  being  removed,  can  be  used  sepa- 
rately or  in  combination  as  simple  magnifiers.] 

[11.  The  Histological  Dissecting  Microscope,  a  combined 
simple  and  compound  microscope  made  by  R.  and  J.  Beck  of 
London*  and  Philadelphia,  figured  in  Plate  XIII,  is  adapted  to 
a  considerable  range  of  family,  school  and  amateur  use.  The 
simple  or  preparing  microscope  figured  at  the  right,  is  small, 
compact  and  substantial ;  and  is  somewhat  suggestive  in  form  of 
the  Zeiss  preparing  microscope.  It  is 'promptly  converted  to  a 
compound  microscope  as  shown  at  the  left,  for  examinations  re- 
quiring higher  powers,  by  removing  the  lens  from  the  transverse 
arm,  and  by  replacing  it  with  a  small  compound  body.  As  a 
simple  microscope  this  instrument  lacks  breadth  of  stage,  and 
as  a  compound,  it  lacks  a  fine  adjustment;  still  it  is  used  with 
much  satisfaction  by  many  whose  wants  do  not  demand  a  higher 
grade  of  apparatus.] 

[12.  Hand  Rests.  In  using  any  form  of  preparing  micros- 
cope, not  even  excepting  those  with  the  largest  stages,  much  in- 
crease both  of  comfort  and  of  steadiness  in  manipulation  can  be 
secured  by  using  hand  rests  at  the  sides  of  the  stage.  Thin 

*  This  instrument,  as  well  as  some  others  mentioned  hereafter,  though  English,  is  ad- 
mitte'd  among  American  apparatus,  for  the  reason  that  the  American  business  relations  of 
the  London  house  have  been  such  for  several  years  past,  that  the  Beck  firm  has  come  to  be 
regarded  as  partly  an  American  enterprise,  and  that  their  wares  have  become  as  familiar 
and  accessible  as  if  actually  made  in  this  country.  R.  H.  W. 


,  ,., 


8. 
8 


bfl 

c 


TJ 

C 
ct 


15 

o 
O 


u 
o 

CD 


THE  PREPARING  MICROSCOPE. 


109 


metal  wings,  as  in  PL  XII,  can  be  attached  to  the  stage  for 
this  purpose.  For  frequent  and  prolonged  use,  however,  a 
broader  and  firmer  support  made  of  wood  and  resting  upon  the 
table  instead  of  the  stage,  is  more  restful  and  is  coming  into 
use.  The  writer  has  been  accustomed  to  use  a  rest,  made 
of  mahogany  strips  about  1cm.  thick,  and  10  to  12  wide,  con- 
structed as  shown  in  front  view,  somewhat  diagraphically,  at 
about  one-fourth  size  in  Fig.  40 ;  where  there  is  a  base  lying 


FIG.  40. 


upon  the  table,  the  rests  attached  at  one  end  by  hinges  and 
held  down  firmly  with  brass  hooks,  hinged  strips  supporting 
the  rests  at  the  desired  height  and  in  an  inclined  position, 
and  wooden  buttons  held  by  large  screws  (which  for  better 
stability  should  be  fastened  with  brass  nuts  below)  for  holding 
the  base  of  the  microscope  firmly  in  position.  The  hinges 
are  all  so  arranged  that  the  strips  can  be  folded  together 
solidly,  for  portability,  as  shown  in  Fig.  41,  and  held  snugly  in 


F 

®  Z        •••::! 

/     \     ^p  _^  ® 

\ 

L     E 

J  1  n  r1!! 

Pn  !!  i  L— 

)         1 

3iUj  1  JJ 

K    1  ii  ]JJV_ 

\  r 

33E    "^ 

•4^"      .ii^ip 

FIG.  41. 

that  position  by  the  same  hooks  as  when  open.  The  hooks 
are  on  the  farther  side  of  the  wooden  strips.  Such  an  arrangement 
can  be  purchased  from  the  microscope  dealers,  or  made  for  one's 
own  use  by  any  person  fond  of  such  experiments.  By  a  slight 
change  in  size  it  is  applicable  to  any  preparing  microscope.  It 
should  be  made  of  such  size  that  the  upper  ends  of  the  rests 


110 


THE  MICROSCOPE  IN  BOTANY. 


will  be  nearly  continuous  with,  or  slightly  below,  the  stage  of 
the  microscope.  Exact  approximation  is  not  necessary.  When 
properly  adjusted  the  rest  is  perfectly  firm  and  steady. 
When  portability  is  not  required,  the  hinges  and  hooks  may 
be  dispensed  with,  and  the  wooden  strips  fastened  together 
with  glue  and  brads.  R.  H.  W.] 


II.     APPARATUS    FOR     DRAWING     MICROSCOPIC 

PICTURES. 

If  we  suppose  that  m,  Fig.  42,  be  the  tube  of  a  microscope,  d 
the  objective  and  c  the  ocular,  then  the  preparation  o  lying  on 


jn 


FIG.  42. 


the  stage  sends  out  a  bundle  of  rays  which,  as  the  mathematical 
line  or,  passes  out  of  the  ocular  in  the  vertical  direct  1311,  or, 
in  the  way  already  described.  Now  place  a  small  glass  mirror  s 
directly  over  the  ocular,  inclined  at  an  angle  of  45°  to  the  emitted 
rays,  and  the  rays  will  be  reflected  at  a  like  angle  from  the  mir- 
ror, changed  now  from  a  vertical  to  a  horizontal  direction.  If 
I  put  my  eye  at  the  point  a,  I  shall  naturally  perceive  the  mi- 
croscopic image  reflected  in  the  mirror.  If  now  the  mirror  be 
transparent  and  a  sheet  of  paper  p  be  fixed  up  perpendicularly 


APPARATUS  FOR  DRAWING  MICROSCOPIC  PICTURES.      Ill 

behind  it,  I  shall  see  through  the  mirror  and  look  upon  the 
paper  beyond  and  the  microscopic  image  will  appear  to  be  ly- 
ing on  the  paper,  or,  in  other  words,  it  will  be  projected 
through  the  mirror  upon  the  paper.  If  we  choose,  the  distance 
[ab  =  10  inches  (25.4  cm.),  the  magnifying  power,  which  is 
really  a  question  of  angular  extent,  will  be  always  converted  to 
linear  measure  at  a  fixed  distance,  as  it  should  and  must  be  to 
render  a  variety  of  records  by  different  observers  comparable 
with  each  other.  The  magnifying  power  represented  by  com- 
paring the  size  of  the  drawing  made  at  this  standard  distance 
with  the  actual  size  of  the  object  itself  will  also  represent  more 
accurately  than  at  any  other  distance  the  resolving  power  of  the 
instrument ;  since  the  power  of  the  microscope  to  render  small 
objects  or  fine  points  of  structure  distinguishable  depends  on 
the  angular  size  of  the  object  as  seen  in  the  microscope  com- 
pared with  that  of  the  object,  not  as  unseen  by  the  naked  eye 
upon  the  microscope  stage,  but  as  it  could  be  seen  by  the  naked 
eye  at  the  best  distance  that  could  be  chosen  for  that  purpose. 
By  common  usage  this  distance  is  established  at  the  standard 
limit  of  10  inches  (25.4cm.)  which  is  assumed  to  be  an  average 
representation  of  the  distance,  varying  for  different  eyes,  of 
most  distinct  vision  for  small  objects.  The  impropriety  of  the 
advice,  which  does  not  lack  high  authority,  to  project  or  draw 
the  magnified  images,  for  measurement  or  comparison,  at  the 
exact  distance  of  the  object  on  the  stage  becomes  evident  in 
such  extreme  cases  as  using  a  simple  microscope,  with  the  object 
very  near  the  eye,  or  a  compound  microscope  having  a  tube  two 
or  three  inches  or  as  many  feet  in  length.  E.  H.  "W.] 

One  may  observe  the  working  of  this  simple  contrivance, 
very  easily  in  the  following  way.  Fix  a  clean  cover-glass 
of  the  utmost  possible  thinness  to  the  top  of  an  ocular,  by  means 
of  a  drop  of  wax,  in  such  a  position  that  it  will  meet  the 
rays  of  light  from  the  objective  at  an  angle  of  45°,  Fig.  42,  s. 
Powerfully  illuminate  the  field  of  view,  and  put  on  a  lo\v  magni- 
fying power  ;  then  the  image  of  the  object  beneath  will  be  seen 
projected,  and  somewhat  darkened,  on  the  paper  b.  With  a 
pointed  lead  pencil,  one  may  then  easily  trace  the  coarser 
outlines  on  the  paper,  since  one  can  see  at  the  same  time  both 


112 


THE  MICROSCOPE  IN  BOTANY. 


the  image  and  the  pencil  point.  This  simple  contrivance  would 
be  perfectly  satisfactory  for  tracing  microscopic  images,  were 
it  not  for  two  faults.  First,  the  paper  surface  is  in  a  very 
unfavorable  position,  the  hand  having  no  support,  hence  the 
tracing  will  be  in  coarse,  rough  outlines,  and  secondly,  the 
image  is  very  poorly  lighted.  Also,  according  to  the  investiga- 
tions of  Fresnoi,  in  the  reflection  of  a  transparent  mirror, 
the  0.944  part  of  all  the  rays  that  fall  upon  the  glass  pass 
through  it  and  only  the  0.056  part  are  reflected  and  come  to  be 
of  value  in  the  reflection-image,  so  that  this  will  have  but  the 
1*8  part  of  the  brightness  of  the  original  image.  These  prevail- 
ing faults  can  be  overcome  in  great  part  in  the  following  way. 

In  order  to  project  the  image  on  a  horizontal  surface  it  is  nec- 
essary [to  place  the  microscope  body  horizontally  while  draw- 
ing, or  else]  to  have  a  contrivance  for  double  reflection.  This  is 

represented  in  Fig.  43,  where 
m again  is  the  microscope-tube 
bent  at  a  right  angle  at  r, 
where  there  is  a  small  thin- 
glass  mirror  s  placed  at  an 
angle  of  45°  to  the  ray  or. 
The  ray  on  striking  this  is  re- 
flected in  the  direction  of  w1 
through  the  ocular  c.  Here 
again  a  transparent  mirror  is 
set  at  an  angle  of  45°  which 
reflects  the  rays  in  the  direction 
FIG' 43<  r'a,  and  the  image  will  appear 

to  the  eye  to  be  projected  on  the  horizontal  surface  beneath  at  5, 
where,  on  the  already  prepared  paper,  the  tracing  may  be  con- 
veniently done. 

One  can  easily  construct  this  contrivance  himself,  by  using  a 
paper  tube  bent  at  r,  at  a  right  angle  and  made  to  fit  on  over  the 
microscope-tube,  and  into  which  an  ocular  can  be  put.  The 
mirror  s,  which  should  be  made  of  the  thinnest  glass,  may  be 
blackened  with  India  ink  on  the  back  side.  By  making  the 
magnification  low,  and  the  light  strong,  this  simple  apparatus  is 
very  well  adapted  to  the  purposes  of  microscopical  drawing.  In 


APPARATUS  FOR  DRAWING  MICROSCOPIC  PICTURES.      113 

order  to  save  the  great  quantity  of  light  which  passes  through 
the  transparent  mirror,  s',  not  reflected,  a  small  metallic  mirror 
is  substituted,  which  not  being  transparent  reflects  all  the  light 
that  falls  upon  it.  It  should  be  made  of  silver,  and  supported 
on  a  very  slender  arm.  It  must  also  be  smaller  than  the  pupil 
of  the  eye,  in  order  that  the  eye  may  look  by  it  and  perceive  at 
the  same  time  the  underlying  paper,  pencil,  etc.,  and  the  image 
have  the  appearance  of  being  projected  upon  the  paper.  Som- 
mering1  invented  the  small  metallic  mirror  as  an  aid  to  micros- 
copical drawing. 

The  use  of  the  reflecting  glass  mirror  has  always  had  this  dis- 
advantage that  it  did  not  give  a  perfectly  sharp  image,  and 
for  this  reason.  We  will  suppose  ABC  D,  Fig.  44,  to  represent 
a  magnified  section  of  a  glass 
mirror  blackened  upon  the  back. 
On  this  mirror  the  light  ray  on 
falls  at  an  angle  of  45°.  Here  a  . 
part  of  the  ray  undergoes  reflection, 
in  consequence  of  which  this  part  of 
the  beam  of  light  takes  the  direc- 
tion n  m  (angle  A  no  =  B  n  m). 
Another  part  of  the  beam,  how- 
ever, passes  through  the  glass 

being  refracted  by  it  in  the  direction  n  n1  to  the  back  side 
of  the  glass.  Here  it  is  reflected  so  that  angle  C  n'n  =  D 

o  o 

n'n".  Here  it  again  suffers  refraction  at  the  front  surface  A  B, 
and  in  consequence  goes  as  a  ray  in  the  direction  71'  m'  parallel 
with  the  ray  reflected  from  the  front  surface  n  m.  By  the  use 
of  the  glass  mirror,  therefore,  the  ray  will  be  divided  into  two 
rays  which  run  near  and  parallel  to  each  other,  and  whose  dis- 
tance apart  will  be  greater  in  proportion  to  the  thickness  of  the 
glass  used.  So  the  image  reflected  from  a  glass  mirror  will  be 
divided  into  two  images  which  are  not  exactly  superimposed, 
and  the  consequence  is,  the  image  is  less  distinct  after  reflection 
than  before. 

This  evil  is  obviated  by  the  use  of  a  reflecting  glass  prism, 
represented   in   section   as   a   right-angled    isosceles    triangle, 

1See  H.  v.  Mohl,  Mikrogvaphie,  p.  321.— Ilarting,  Das  Mikroskop,  pp.  176,  901. 
8 


114 


THE   MICROSCOPE  IN  BOTANF. 


which  makes  the  reflection  from  the  hypothenuse.  The  latter 
must  be  ground  absolutely  flat,  which  is  by  no  means  an 
easy  thing  to  do.  Fig.  45  represents  two  reflecting  glass 
prisms  which  are  so  arranged  as  to  correspond  to  the  two  glass 

mirrors  in  Fig.  43,  r  and  r'  in 
both  figures  corresponding  to  each 
other.  We  will  suppose  that  the 
ray  o  passing  in  the  direction  of 
the  arrowpoint  falls  upon  the  side 
of  the  prism  p  perpendicular  to  its 
surface.  It  enters  the  glass  unre- 
fracted  and  passes  to  r,  striking  the  hypothenuse  surface  at 
an  angle  of  45°.  Here  it  suffers  a  total  reflection  and  takes 
the  direction  rr'.  In  like  manner  it  enters  the  second  prism 
p'  and  is  likewise  reflected  at  rf  in  the  direction  r'a.  It  is 
therefore  clear  that  the  two  mirrors  s  and  s',  Fig.  43,  can 
.be  replaced  by  the  prisms  pp'. 


TIG.  45. 


FIG.  45. 

[A  prism  like  p',  but  about  half  the  size  figured,  is  sometimes 
cemented  to  the  end  of  a  small  bar  and  mounted  in  front  of  the 
ocular,  the  large  prism  being  dispensed  with.  It  is  very  con- 
venient for  drawing  either  in  the  horizontal  or  inclined  position 
of  the  tube ;  but  as  it  inverts  the  image,  as  do  other  singly- 


APPARATUS  FOR  DRAWING  MICROSCOPIC  PICTURES.       115 

reflecting  arrangements,  difficulty  is  experienced  in  retouching 
the  sketch  made  by  its  use.  It  also  requires  great  steadiness  of 
position  on  the  part  of  the  user.  By  substituting  for  the  little 
prism  a  small  steel  mirror,  also  smaller  than  the  pupil  of  the 
eye,  the  Sommering  mirror  is  produced,  which  acts  precisely 
like  the  small  prism  and  has  much  the  same  advantages  and 
defects.  Neither  of  the  above  has  come  into  very  general  use.] 

[1.  The  Neutral  Tint  Reflector.  The  singly-reflecting  cam- 
era, called  the  neutral  tint  reflector,  was  brought  into  use  at  the 
suggestion  of  Dr.  Beale  of  London  and  is  shown  in  situ  in 
Fig.  46.  This  reflector  was  originally  a  thin  plate  of  neutral 
tint  glass,  but  a  common  white  cover-glass  is  now  employed 
as  a  satisfactory  substitute.  It  is  supported  in  front  of  the 
ocular  at  an  angle  of  45°,  so  that  the  observer  looks  ob- 
liquely through  it  at  the  paper  and  pencil,  seeing  at  the 
same  time  and  apparently  in  the  same  place  the  microscopic 
image  reflected  from  the  glass.  The  glass  being  thin,  but  little 
indistinctness  results  from  the  confusion  of  the  separate  reflec- 
tions from  its  two  surfaces;  while  the  inconvenience  of  its 
inverting  the  image  seems  to  have  been  quite  overbalanced  by 
its  simplicity  and  cheapness,  and  the  fa- 
cility with  which  it  can  be  used  even  by 
inexpert  persons.  Though  practically 
limited,  for  the  reason  stated  above,  to 
those  uses  which  allow  a  nearly  horizontal 
position  of  the  microscope  body,  this  con- 
trivance is  probably  more  used  in  this 
country  than  any  other  form  of  camera 
lucida.  R.  H.W.] 

2.  The  Wollaston  Camera  lucida.  The 
little  mirror  s  in  Fig.  42,  s'  in  Fig.  43, 
and  the  prism j/  in  Fig.  45  may  be  replaced 
hy  an  apparatus  invented  by  Wollaston 
and  represented  in  Fig.  47. 2  It  consists  of  a  four  sided  glass 

2  See  Wollaston  in  Phil.  Transactions,  1809,  No.  38,  p.  741.  —  W.  H.  Wollaston's  descrip- 
tion of  the  camera  lucida,  an  instrument  designed  for  sketching  objects  in  the  neighbor- 
hood, and  for  making  magnified  or  minified  tracings  (Gilbert's  Annalen  der  Pliysik.  Bd. 
XXXIV.  N.  F.  Bd.  IV,  1S10,  pp.  353-361,  I  Tafel)  —  Gehler's  Physikalisches  Worterbuch, 
Leipzig,  1825,  Bd.  II,  p.  30^. 


116  THE  MICKOSCOPE  IN  BOTANY. 

prism  AFGH,  in  which  FGH  is  a  right  angle  while  HAF 
equals  135°.  The  ray  strikes  the  surface  GF  in  the  vicinity 
of  F  perpendicularly,  so  that  it  passes  unreflected  through 
the  prism  to  r  and  then  to  r'  in  each  undergoing  total  reflec- 
tion, and  passing  out  of  the  prism  in  the  direction  r'a.  The 
eye  placed  at  the  point  a  looks  downward  through  and  just 
beyond  the  edge  of  the  prism  at  H,  and  perceives  the  sheet 
of  paper  lying  at  p,  on  which  at  b  the  image  of  the  rays  seems 
to  be. 

[The  Wollaston  camera  lucida  is  usually  attached  to  the  mi- 
croscope by  means  of  a  spring-ring  slipping  over  the  top  of  the 
ocular.  Connected  with  the  front  end  of  this  ring  is  a  light 
brass  box  containing  the  prism  and  wholly  covering  it  except 
that  the  side  FG  in  Fig.  47,  is  left  unprotected  and  that  the 

edge  His  exposed  (more  clearly  seen 
at  F,  Fig.  48),  by  a  notch  through 
which  the  observer  looks  down  upon 
and  through  the  prism,  at  the  same 
time  that  he  views  the  paper  and 
pencil  with  that  portion  of  the  pupil 
of  the  eye  that  is  not  over  the  prism. 
This  camera  lucida,  originally  devised 
for  general  drawing,  is  still  used  more 

than  almost  any  other  for  microscopical  work.  Care  is  re- 
quired as  in  the  use  of  other  cameras,  to  so  regulate  the 
intensity  of  the  illumination  of  the  field  of  view  and  of  the  draw- 
ing paper  that  neither  shall  be  obscured  by  the  relative  bright- 
ness of  the  other.*  For  greater  ease  and  distinctness  in 
viewing  the  paper,  a  lens  of  long  focus,  like  a  spectacle  glass, 
is  often  placed  below  the  prism,  just  below  ?•',  Fig.  47,  and  in 
the  line  of  the  ray  ab.  This  camera  is  generally  used  with  the 
body  of  the  microscope  in  a  horizontal  position,  and  with  the 
paper  lying  horizontally  beneath  the  ocular,  since  a  vertical 

*  As  the  light  in  the  field  of  view  and  on  the  object  may  be  easily  and  exactly  regulated 
by  means  of  the  diaphragm  and  mirror,  the  principal  difficulty  will  be  found  in  the  man- 
agement of  the  light  upon  the  paper.  Hence  a  small  movable  screen  of  thick  paper,  which 
one  may  easily  contrive  lor  himself,  about  30  cm.  long  and  20  cm.  high,  placed  more  or  less 
directly  between  the  source  of  light  and  the  paper,  thus  darkening  the  latter  at  will  with 
its  shadow,  will  be  found  very  useful  in  conducting  this  kind  of  microscopical  drawing. 
A.  B.  H. 


APPARATUS  FOR  DRAWING  MICROSCOPIC  PICTURES.        117 


position   of   the   tube   would    require    the   paper   also   to   be 
vertical.     R.  H.  W.] 

3.  NoberCs  Camera  lacida.  Another  camera  lucida  origi- 
nated with  Nobert  and  is  diagrammatically  represented  in  Fig. 
49.  It  permits  the  picture  to  be  drawn  on  a  horizontal  surface. 
The  rhombic  prism,  ABCD,  bears  on  its  oblique  surface  AB 
a  small  right  angle  prism  EFG,  made  fast  to  the  surface  AB 
at  EF  by  means  of  Canada  balsam.  It  is  then  so  placed  over 
the  ocular  of  the  microscope  that  a 
ray  of  light  proceeding  from  it 
strikes  the  surface  FG  perpendicu- 
larly. It  goes  directly  through  the 
mass  of  glass  to  a  in  the  direction  oa, 
vmrefracted  and  losing  little  light  by 
reflection  at  rr.  Again  ray  b  from 
a  horizontal  drawing  surface  strikes 
the  oblique  side  BC  of  the  rhombic 
prism  and  passes  through  to  the  side 
total  reflection  in  the  direction  r 


T) 


1 


A 


C 


1. 


FIG.  49. 


CD  at  r  and  suffers  a 
At  ^  it  is  again  totally 
reflected  in  the  direction  ra,  and  reaches  the  observer's  eye  at 
the  same  time  and  in  the  same  direction  with  the  image-forming 
rays  from  the  microscope  oa.  The  observer  sees  in  the  micro- 
scope, by  means  of  this  apparatus,  not  only  the  object  but  at 
the  same  time  also  an  image  of  the  drawing  surface  and  pencil. 

The  Nobert  draw- 
ing prism  as  com- 
pleted by  Nachet 
[and  known  in  this 
country  as  Nachet's] 
is  represented  natu- 
ral size  in  Fig.  50. 
This  apparatus  for 
horizontal  d  r  a  w  i  n  g 
consists  of  a  metal 
ring  r  which  is  put  upon  the  microscope-tube  and  the  ocular 
is  replaced.  The  metal  ring  bears  a  projection  h,  into  which 
the  brass  rod  f  exactly  fits,  and  not  only  turns  upon  its  axis 
but  likewise  may  be  moved  up  and  down  by  hand.  On  f 


FIG.  50. 


118 


THE  MICROSCOPE  IN  BOTANY. 


is  fastened  a  blackened  metal  plate  cZ,  which  widens  into  a  circle 
over  the  ring  and  has  its  center  bored  out  with  a  large  opening. 
The  metal  box  AB  is  fastened  to  this  plate  by  means  of  the 
screws  e.  The  box  contains,  within,  the  two  glass  prisms  more 
exactly  illustrated  in  Fig.  49.  At  C  and  in  the  corresponding 
place  of  the  under  surface  is  a  circular  opening  which  falls  in 
with  that  of  the  plate  d  just  now  mentioned.  The  box  must  be 
so  large  that  B  shall  extend  laterally  beyond  the  foot  of  the  mi- 
croscope. Directly  under  B  is  the  surface  of  the  paper  on 
which  the  drawing  is  to  be  done.  The  eye  looks  down  through 
G  and  sees  in  the  field  of  the  microscope  the  drawing  paper  and 
the  pencil.  The  box  AB  may  be  turned  aside  at  will  and  leave 
the  ocular  free. 

[4.  Grunow's  Camera  lucid  a. 
Mr.  J.  Grunow  has  contrived  a 
camera  lucida  in  which  an  effect 
nearly  identical  with  that  of 
Nobert  and  Nachet  is  secured, 
but  in  a  slightly  different  manner. 
This  device  is  shown  in  section 
in  Fig.  51  where  P  is  a  rectangu- 
lar reflecting  prism  like  those  in 
Fig.  45,  while  P'  is  a  cube  formed 
of  two  such  prisms  cemented 
together  with  Canada  balsam. 
One  of  the  surfaces  of  contact, 
fg,  is  silvered,  except  a  circular 
spot  in  the  center  about  half  the 
diameter  of  the  pupil  of  the  eye,  thus  bisecting  the  cube 
obliquely  with  a  perforated  mirror.  It  is  evident  that  the  eye 
at  PN  can  look  directly  down  the  microscope-tube  in  the 
direction  NM  through  the  cube  P'  by  reason  of  the  central 
aperture  in  the  silvered  surface  fg,  while  the  paper  on  the 
table  at  P  can  be  seen  with  ease  at  the  same  time  by  re- 
flection from  the  glass  surface  P  and  from  the  silvered  portion 
of  the  interior  surface  fg.  This  device  is  mounted  and  slipped 
over  the  ocular.  It  is  used  with  the  same  comfort  to  the 
eyes  as  the  Nobert  form,  though  not  with  the  same  position 


FIG.  si. 


APPARATUS  FOR  DRAWING  MICROSCOPIC  PICTURES.       119 

of  the  microscope,  since  the  Grunow  camera  is  best  adapted 
to  an  inclined  position  of  the  microscope-tube,  making  the  line 
PP  vertical  and  the  drawing  upon  the  table  free  from  distortion 
when  the  tube  is  inclined  at  about  an  angle  of  30°,  while 
Nobert's  gives  the  same  results  in  a  vertical  position  of  the  mi- 
croscope, and  requires  an  inclined  drawing  board  when  the  tube 
is  inclined.] 

[5.  Photo-micrography.  The  substitution  of  the  camera  ob- 
scura  for  the  camera  lucida  as  a  means  of  preserving  or  repro- 
ducing microscopic  views,  while  not  without  disadvantage  in 
respect  of  showing  the  relation  of  parts,  and  of  selecting  typical 
points  in  different  portions  of  the  field  and  combining  them  all 
in  one  picture,  is  often  desirable  on  account  of  the  impartiality 
and  completeness  of  detail  secured  by  the  automatic  action  of 
the  light  itself.  Hitherto  its  use  has  been  mostly  confined  to 
the  few  who  happened  to  possess  an  exceptional  access  to,  and 
familiarity  with,  the  mysteries  behind  the  scenes  of  some  photo- 
graph gallery,  and  to  the  still  smaller  number  who  were  pre- 
pared to  incur  the  expense  of  employing  the  assistance  of  a 
professional  photographer.  Recently,  however,  the  develop- 
ment of  amateur  photography  as  a  popular  pastime  has  placed 
within  reach  of  the  microscopist  the  means  of  doing  this  work  for 
himself,  without  previous  experience,  unusual  mechanical  skill, 
or  considerable  expense.] 

[The  Bausch  and  Lomb  Optical  Co.  offer  for  sale  a  very 
compact  and  beautiful  amateur  photographic  camera,  suitable 
for  general  work,  with  a  simple  attachment  by  means  of  which 
it  can  be  brought  into  relation  with  the  possessor's  microscope, 
and  the  magnified  image  of  an  object  focussed  upon  its  sensi- 
tized plate.  Mr.  W.  H.  Walmsley  of  Philadelphia,  late  Am- 
erican Manager  for  the  Messrs.  Beck,  has  arranged,  and  has 
introduced  to  the  trade,  a  complete  outfit,  containing  not  only 
a  camera,  and  platform  for  connecting  with  the  microscope,  but 
also  the  requisite  chemicals,  and  a  complete,  carefully  selected 
assortment  of  the  various  supplies  required  for  the  work.  This 
will  be  a  convenience  to  those  who  do  not  wish  to  incur  the 
trouble  and  delay  of  learning  their  own  wants  by  means  of  their 


120 


THE  MICROSCOPE  IN  BOTANY. 


own  experience.  Mr.  Walmsley's  arrangement  of  his  apparatus 
is  shown  in  Fig.  52,  which  illustrates  clearly  the  theory  and 
practice  of  such  devices  generally.  R.  H.  W.] 


FIG.  52. 


III.  THE  MICROMETER  AND  MICROSCOPICAL 
MEASURING. 

In  microscopical  investigations  it  is  often  a  matter  of  great 
importance  to  be  able  to  determine  the  true  size  of  an  object 
under  the  microscope.  This  can  be  done  in  various  ways.  But 
as  different  as  the  various  apparatus  is  which  has  been  designed 
for  this  purpose,  there  are  but  two  principles  involved  in  it, 
viz.,  either  to  measure  the  object  itself,  or,  to  measure  the  mag- 
nified image  of  it  which  the  objective  produces.  The  appara- 
tus which  aims  to  do  the  former,  we  call  "objective  micrometers  :" 
that  which  aims  to  accomplish  the  latter  we  name  "ocular  mi- 
crometers." Objective  as  well  as  ocular  micrometers  may  be 
either  glass-  or  screw-micrometers,  that  is  their  essential  part 
may  be  fine  diamond-rulings  on  glass,  or  a  carefully  cut  screw 
\vhose  thread  has  a  definite  height. 


OBJECTIVE  MICROMETERS.  121 


I.    OBJECTIVE  MICKOMETEKS. 

A.  The  Objective  Glass- Micrometer.     (Stage  micrometer.) 
This    consists   of  a  small  glass  plate  on  which,  by  means  of  a 
diamond,  fine  rulings  are  cut,  a  millimeter  being  divided  into  one 
hundred  equal  pails.      [Its  appearance  under  the  microscope  is 
as  represented  in  Fig.  53.     Subdivisions  of  the  inch,  or  inch 
and  millimeter  compared,  are  also  used.]     If  such  a  ruled  plate 
be  put  under  the  microscope  in  place  of  the  object  slide  and  the 
object  to  be  measured  be  laid  upon  it,  the  length  of  the  latter  can 
be  determined.    This  kind  of  microscopical  measuring  is  doubt- 
less the  simplest  in  the  world,  but,  alas  !  there  come  in  so  many 
disturbing  influences  that  its  use  is  limited  to  a  few  cases  only. 
[It  is  chiefly  used  to  determine  the  working  value  of  the  divi- 
sions of  the  ocular  micrometer.] 

B.  The  Objective  Screw-Micrometer.     [This  apparatus  is  a 
sliding  plate  attached  to  the  stage,  or  constituting  a  part  of  the 
stage  itself,  by  which  the  object  while  in  view  in  the  micros- 
cope  is  carried   steadily  past  a   fine   line  fixed  in  the  ocular. 
The  movement  is  accomplished  by  the  push  of  a  fine  horizontal 
screw   acting   upon  'the   plate,  and  the  distance  traversed    in 
carrying  the  whole  object  across  the   line,  the  same  being  the 
diameter  of  the  object  itself,  is  determined  by  the  number  of 
the  turns  and  fractions  of  a  turn  of  the  screw,  the  width  of  whose 
thread  is  very  accurately  known.     Though  manufactured  with 
great  skill,  and  theoretically  capable  of  reading  off  results  with 
almost  unlimited  minuteness,  directly  and  without  the  trouble 
of  computations  pertaining  to  ocular  micrometers,  this  apparatus 
requires  great  skill  in  manipulation  and  is  particularly  liable  to 
get  out  of  order.     At  best  it  is  believed  to  be  inferior  to  the 
ocular  micrometer  in  accuracy,  since   it   measures    the   object 
directly,  and  not  its  magnified  image  ;  any  errors  in  its  perform- 
ance being,    therefore,  multiplied    by  the  whole    magnifying 
power  of  the  microscope,  instead  of  by  the   power  of  the  eye 
lens  only.     For  these  reasons  it  is,  as  conceded  by  the  author, 
giving  place  more  and  more  to  the  ocular  micrometer.] 


122  THE  MICROSCOPE  IN  BOTANY. 

[Some  microscopes  with  a  mechanical  stage  have  the  adja- 
cent surfaces  graduated,  with  or  without  a  vernier,  so  that  the 
position  of  the  stage  or  the  distance  it  has  moved  can  be  read  off 
and  recorded.  This  serves  as  a  finder,  by  which  the  position 
of  an  object  mounted  on  a  specified  slide  may  be  registered, 
and  supplies  a  means  of  roughly  measuring  the  size  of  large  ob- 
jects, as  for  instance,  the  width  of  a  leaf  or  the  length  of  an 
anther.  R.  H.  W.] 


2.    THE  OCULAR  MICROMETER. 

Ocular  micrometers  by  which  we  undertake  to  measure  the 
real  image  of  the  object  are  either  glass-  or  screw-micrometers. 
They  have,  however,  this  advantage  over  the  objective  microm- 
eter, that  they  do  not  require  by  far,  the  same  precision  of  work 
as  that,  since  a  casual  error  in  the  rulings,  etc.,  has  a  mag- 
nitude in  the  results  of  the  measurement,  equal  only  to  the 
given  .  error  divided  by  the  whole  number  of  the  objective 
magnification. 

A.  Ocular  Glass-Micrometer.  This  apparatus  has  a  wider 
distribution  in  Germany  than  all  other  micrometers.  It  con- 
sists, as  the  objective  glass-micrometer  does,  of  a  glass  plate 
upon  which  are  diamond-rulings.  A  thin  cover-glass  is  put 
over  the  rulings  for  their  better  protection.  The  scale  extends 
for  four  or  five  mm.,  and  each  millimeter  is  divided  into  ten  or 
twenty  equal  parts,  the  fifth  and  tenth  line  being  extended  in 
the  ordinary  way.  for  greater  convenience  in  counting. 

[Whether  it  be  divided  into  metric  or  English  units  is  unim- 
portant, except  that  it  may  sometimes  be  required  for  use  as  a 
stage  micrometer,  since  the  working  value  of  the  divisions  de- 
pends upon  the  optical  conditions  under  which  they  are  used, 
and  must  be  carefully  determined  after  the  apparatus  is  arranged 
and  in  order  for  the  proposed  work.] 

[The  simplest,  cheapest  and  commonest  form  of  ocular  microm- 
eter is  a  round  cover-glass,  properly  ruled,  which  is  laid  when 
about  to  be  used  upon  the  diaphragm  of  the  ocular  in  the  focus 
of  the  eye  lens.  The  rulings  should  be  turned  downwards  to 


THE  OCULAR  MICROMETER.  123 

bring  them  close  to  the  diaphragm,  and  if  not  distinctly  visible, 
they  should  be  made  so  by  slightly  unscrewing  the  lens,  and  thus 
bringing  them  into  its  focus.  When  properly  adjusted,  the  ruled 
scale  will  appear  clearly  defined  in  the  field  of  view  somewhat  as 
represented  by  Fig.  53.  Sometimes  the  glass  is  cut  away  at 

one  end  of  the  lines,  leaving 
them  at  the  edge  of  a  half-cir- 
cular disk ;  and  thus,  without 
impairing  the  micrometer,  half 
the  field  is  presented  with  its 
definition  undisturbed  by  the 
additional  glass  plate.  Some 
makers  mount  the  glass  scale  in 
a  plate  of  brass  or  hard  rubber 
to  be  slipped  into  the  ocular  just 
above  the  diaphragm  through 
FIG-  53'  slits  left  for  that  purpose ;  the 

slits  being  closed  by  a  short,  sliding  tube,  when  the  micrometer 
is  not  in  use.  Unless  a  screw  be  attached,  as  is  sometimes 
done,  giving  a  delicate  lateral  movement,  the  use  of  any 
ocular  micrometer  is  much  -facilitated  by  the  employment 
of  the  mechanical  stage,  by  means  of  -which  a  coincidence  of 
certain  lines  Avith  any  part  of  the  object  to  be  measured  can 
be  the  better  secured.  K.  H.  W.J 

Measuring  by  means  of  the  ocular  micrometer  is  conducted 
in  the  following  way.  The  micrometer  is  put  in  place,  its  scale 
and  the  object  brought  in  focus,  and  then  by  turning  the  ocular 
and  moving  the  object,  the  whole  length  of  the  object  to  be 
measured  is  covered  with  the  scale,  in  such  a  way  that  its  rulings 
are  perpendicular  to  the  longer  axis  of  the  object.  Then  adjust 
the  object  as  sharply  as  possible  and  count  up  how  many  of  the 
rulings  are  covered  by  it  (whole  and  fractions) .  This  number 
is  to  be  noted  down  and  if  one  desires  very  great  exactness  the 
measurement  may  be  repeated  on  different  parts  of  the  scale 
and  the  results  averaged. 

It  is  clear  enough  that  one  does  not  get  the  exact  and  real 
size  of  the  object  by  this  process.  To  get  this  one  must  either 
exactly  determine  the  number  of  magnifications  of  the  objective- 


124  THE  MICROSCOPE  IN  BOTANY. 

system  together  with  that  of  the  field  glass  and  the  eye  lens,  so 
as  to  ascertain  the  true  value  of  the  ruled  spaces  of  the  microm- 
eter, or  he  must  determine  their  true  value  by  the  use  of  an 
exact  objective  glass-micrometer.  If  he  has  for  instance  found 
that  in  tbe  first  case,  the  objective  and  the  field  lens  together 
magnify  seventy  diameters  and  the  rulings  of  the  micrometer 
are  0.05  mm.  apart,  so  by  the  use  of  that  objective-system 
the  space  between  two  rulings  will  amount  to  a  magnitude  in  the 
object  of  0.714  of  a  micromillimeter.  The  determination  of 
the  relative  value  of  the  ocular  micrometer  by  means  of  the 
objective  glass-micrometer  is  very  simple.  The  latter  is  used 
as  an  object,  and  then  it  is  determined  how  many  of  its  divisions 
answer  to  a  certain  number  of  the  divisions  of  the  ocular  micro- 
meter. Divide  the  first  by  the  last  and  the  result  will  be  the 
true  value  of  the  ocular-micrometer  divisions  in  units  of  the  ob- 
jective micrometer.  If,  for  instance,  20  divisions  of  the 
ocular  micrometer  cover  an  extension  of  87  micromillimeters  of 
the  objective  micrometer,  the  value  of  an  interval  in  the  ocular 
micrometer  will  be  f  &  =  4.35^  =  0.00435  mm.  [In  such  work, 
round  numbers  are  always  preferred,  for  the  sake  of  simplify- 
ing the  labor  -of  computation  and  comparison.  In  the  above 
example,  for  instance,  the  20  divisions  of  the  ocular  micrometer 
could,  by  lengthening  the  optical  axis  of  the  microscope  by 
slightly  raising  the  draw-tube,  be  made  to  cover  100  micro- 
millimeters  instead  of  87,  the  value  of  one  interval  becoming  5/j. 
instead  of  4.35,u,  and  the  labor  of  computation  being  reduced 
to  almost  nothing.  So  in  the  author's  following  illustration, 
the  values  8,  3.63,  1.27,  0.55  would  by  a  judicious  use  of  the 
draw-tube  be  changed  to  8,  4,  1.5  or  even  2,  0.6  or  even  1 
respectively.  Having  once  found  a  convenient  position  of  the 
draw-tube  for  a  certain  purpose,  much  trouble  will  be  saved  by 
recording  that  position  so  that  it  can  be  promptly  restored  when 
next  wanted.  The  draw-tube  should  be  graduated  for  this  pur- 
pose. Having  been  set  in  position,  approximately,  by  means 
of  the  graduation,  a  slight  readjustment  will  instantly  secure 
exact  apposition  of  the  desired  lines  (say  20  to  100)  of  the 
ocular  and  objective  scales.  R.  H.  W.] 

If  the  manufacturer  would  take  pains  to  determine  the  micro- 


THE  OCULAR  MICROMETER.  125 

metric  value  for  every  system  furnished  it  might  be  designated 
[approximately,  but  not  with  sufficient  accuracy  for  fine  work] 
on  the  tables  of  magnification  which  accompany  the  microscope. 
If  this  were  given,  as  we  may  suppose  for  illustration,  for  the 
systems  I,  II,  III,  IV,  of  an  instrument  8.00,  3.63,  1.27,  0.55, 
respectively,  then  I  have  these  numbers  by  which  to  multiply 
the  number  of  units  read  off  from  my  ocular  micrometer  in  order 
to  determine  the  true  size  of  the  object  measured  in  micromilli- 
naeters. 

Thus,  the  object  being  measured  is  found  to  cover  17  divisions 
of  the  ocular  micrometer,  the  magnification  being  with  the  sys- 
tem I.  It  has  consequently  a  true  length  of  17  X  8.=  136,^  ;  or, 
the  magnification  is  made  by  system  IV,  the  object  covering 
53.5  divisions  of  the  micrometer.  Its  true  size  will  then  be 
53.5x0.55=  29.4,*  =  0.0294  mm. 

Some  firms  have  lately  furnished  adjustable  ocular  glass-mi- 
crometers, which  are  provided  with  a  fine  screw  by  means  of 
which  the  rulings  may  be  moved  in  a  horizontal  direction,  and  a 
further  adjustment  by  which  the  rulings  may  be  brought  very 
exactly  iiito  the  focus  of  the  ocular  lens.  For  fine  measurements 
this  is  decidedly  preferable  to  the  one  previously  described. 

[Objective  glass-micrometers,  here  called  stage  micrometers, 
if  based  upon  the  English  units  are  commonly  ruled  to  1,  A 
and  -JL  inch.  The  metric  (decimal)  system,  however,  is  com- 
ing into  somewhat  general  use  here,  and  it  must  be  admitted 
that  1  mm.  (about  JL  in.)  _L  and  ±  mm.  form  a  more  convenient 
series  than  any  round  fractions  of  the  inch,  i  mm.  is,  more- 
over, equal  to  10;-*,  the  micron  or  micromillimeter,  /-*,  being  the 
only  recogized  unit  in  the  world  for  a  minimum  unit  in  meas- 
urements. At  present,  the  metric  values  being  not  yet  familiar 
to  all  students,  stage  micrometers  are  often  ruled  showing  the 
metric  and  English  scales  in  comparison  with  each  other.  The 
stage  micrometer  should  be  ruled  with  the  utmost  possible 
precision,  as  its  errors  are  multiplied  by  the  whole  magnifying 
power  of  the  microscope  employed.  It  has-  been  known  for 
years  that  the  best  micrometers  in  use  contained  perceptible 
errors.  The  standard  micrometer  of  the  National  Committee, 
adopted  in  1883  by  the  American  Society  of  Microscopists, 


126  THE  MICROSCOPE  IN   BOTANY. 

represents  1  cm.  subdivided  to  1,  —  and  ^L  mm. ;  and  the 
value  of  its  spaces,  as  related  to  larger  standards  of  length, 
have  been  determined  with  a  certainty  and  precision  not  known 
to  have  been  attained  before  in  any  micrometric  standard. 
Copies  of  this  standard  can  now  be  obtained  from  the  dealers  in 
microscopes  ;  and  the  loan  of  officially  certified  copies,  for  com- 
parison, can  be  obtained,  under  certain  restrictions,  from  the 
officers  of  the  society.  R.  H.  W.] 


B.    THE  OCULAR  SCREW-MICROMETER. 

This  micrometer  is  seldom  used  in  Germany  but  frequently 
in  England,  where,  however,  on  the  whole,  very  little  and  very 
indifferent  microscopical  research  is  made,  except  indeed,  to 
look  at  diatom  frustules,  and  to  take  delight  in  their  markings ; 
but  for  that,  one  has  there  the  most  expensive  and  showy  ap- 
paratus.* 

The  measuring  apparatus  in  question  differs  very  little  from 
the  objective  screw-micrometer.  By  means  of  a  micrometer 
screw  whose  drumhead  is  divided  into  one  hundred  parts,  a 
movable  thread  is  carried  through  the  field  of  a  Ramsden  ocu- 
lar toward  another  thread  parallel  to  it.  The  revolutions  are 
read  off  from  a  metal  scale  in  the  field  of  the  ocular. 

[This  apparatus,  borrowed  from  the  telescope,  and  familiarly 
known  as  the  "  Ramsden"  or  "  cobweb  "  micrometer,  requires  to 
be  well  made,  and  to  be  attached  to  a  sufficiently  firm  stand. 
It  is  also  rather  expensive.  It  is  believed,  however,  to  be  cap- 
able of  performing  a  series  of  measurements  with  a  rapidity  and 
precision  not  easily  attained  by  any  other  means.  Like  other 
ocular  micrometers,  it  is  most  readily  used  upon  a  micros- 
cope having  a  mechanical  movement  to  the  stage.] 

[Fig.  54  shows  a  cobweb  micrometer  as  now  constructed  by 

*  The  preceding  remark  is  retained  out  of  respect  to  the  author's  liberty  of  opinion,  and 
as  a  very  vivid  caution  (which  might  be  useful  also  in  this  country)  against  a  real  and  con- 
ceded  evil;  but  with  a  very  decided  belief  on  the  part  of  the  writer,  that  the  distinguished 
author's  information  must  have  come  from  such  partial  or  prejudiced  sources  as  to  lead 
him  to  somewhat  overstate  the  faults  of  his  neighbors,  and  to  greatly  underestimate  the 
really  scientific  work  which  is  being  done  in  England.  R.  H.  W. 


THE  CAMERA  LUCIDA  AS  A  MEASURING  APPARATUS.       127 

Mr.  Zentmayer.  It  is  of  convenient  model  and  excellent  work- 
manship, and  is  combined,  very  advantageously,  with  a  gonio- 
meter attachment  represented  in  the  cut  by  the  large  graduated 
circle  with  vernier.  R.  H.  W.] 


FIG.  54. 


III.  THE  CAMERA  LUCIDA  AS  A  MEASURING 
APPARATUS. 


Almost  all  the  contrivances  described  on  pp.  114-118, 
may  be  directly  applied  to  the  measuring  of  microscopic  ob- 
jects. If  one  has  traced  the  exact  outline  of  the  object,  and 
knows  exactly  how  many  times  the  image  has  been  magnified, 
it  is  only  necessary  to  measure  the  drawing  with  the  metric  rule 
and  divide  the  same  by  the  number  of  the  magnifications,  to  get 
the  true  s^:e  of  the  object  in  fractions  of  a  millimeter. 


128  THE  MICROSCOPE  IN  BOTANY. 

It  is  first  of  all  necessary,  in  this  case,  to  determine  with  ex- 
actness the  magnification  which  the  camera  lucida  gives  with 
the  objective  employed.  This  may  be  done  in  the  following 
way.  Set  the  camera  lucida  on  the  microscope  and  use  an  ob- 
jective glass-micrometer  for  an  object  on  the  stage,3  illuminate 
it  with  excentric  light  so  as  to  bring  its  markings  out  clearly, 
and  draw  a  number  of  the  markings  on  a  piece  of  paper  [at  a 
distance  of  10 inches,  25.4  cm.,  from  the  eye,  and  in  a  plane  at 
right  angles,  to  the  line  of  vision].  This  may  be  done  in  this 
way.  Draw  a  straight  line  with  India  ink  across  the  paper. 
Place  this  line  so  that  it  will  lie  exactly  perpendicular  to  the  rul- 
ings of  the  micrometer.  Then  with  a  sharp  pencil  mark  the  points 
of  intersection  of  the  micrometer  rulings  with  the  line,  and  as 
the  lines  by  magnification  have  a  considerable  size,  mark  the 
same  edge  of  each,  right  or  left.  The  markings  in  the  middle 
of  the  field  should  be  selected  for  this  purpose.  Then  ascertain 
the  distance  between  any  two  points  on  the  line  by  the  milli- 
meter scale  ;  repeat  this  for  a  considerable  number  of  the  mark- 
ings, and  getting  the  arithmetical  average,  divide  this  by  the 
micrometer  unit.  The  quotient  gives  the  number  of  the  magni- 
fications. If  one  draws  an  object  on  paper  intended  for  subse- 
quent measurement,  it  should  naturally  be  placed  at  the  [stand- 
ard distance  of  distinct  vision,  viz.  10  inches  =  25.4  cm.  See 
p.  111].* 

IV.     CONCERNING  MICROMETRIC  MEASUREMENTS 
IN  GENERAL. 

In  the  descriptions  of  measuring  apparatus  we  have  here  and 
there  dropped  hints  as  to  their  management.  Some  remarks 
of  a  general  nature  may  not  be  out  of  place  here.  Measurements, 
made  by  the  very  best  micrometers,  only  approximately  express 
the  absolute  size  of  the  measured  object.  The  reason  for  this 

3  If  one  has  no  stage  micrometer  he  may  resort  to  a  good  eye-piece  micrometer,  1mm. 
divided  into  twenty  parts  and  boldly  make  use  of  that.  It  gives,  especially  when  several 
measurements  are  combined,  perfectly  satisfactory  results. 

*  If  one  has  a  microscope  with  a  draw-tube,  he  can  very  easily,  with  his  objective  and 
camera,  produce  a  magnification  of  any  desired  round  number,  which  is  very  handy  in 
simplilying  the  calculations. 


MICROMETRIC  MEASUREMENTS  IX  GENERAL.  129 

is  first  of  all  in  the  fact  that  the  exact  focussing  of  the  object  is 
attended  with  the  very  greatest  difficulty.  Each  worker  here  fol- 
lows his  own  subjective  judgment,  andean  follow  no  other.  He 
works  with  his  eyes  and  his  hands.  When  two  trained  micros- 
copists  focus  the  same  object  under  the  same  microscope,  it  may 
be  assumed  as  a  matter  of  course  that  the  two  adjustments  will 
turn  out  to  be  different.  Indeed,  should  this  not  be  the  case, 
we  must  consider  it  to  be  purely  accidental.  That  measure- 
ments made  by  each  of  the  two  adjustments  must  differ  goes 
without  saying,  and  therefore  it  comes  about  that  measurements, 
made  by  two  different  persons,  can  be  compared  as  to  their  ab- 
solute value  only  with  the  greatest  difficulty.  For  this  reason 
all  those  measurements  which  are  thought  to  be  so  extraordi- 
narily accurate,  and  whose  possibility  was  once  so  long  and  so 
widely  discussed,  become  quite  or  entirely  valueless.  But  most 
objects  which  are  to  be  microscopically  measured  —  organic 
fol-ms  —  appear  under  very  unlike  dimensions  and  it  must  be  a 
matter  of  great  indifference  to  us  whether  the  long  diameter  of  a 
grain  of  starch  from  a  potato,  be  given  ^  too  large  or  too  small. 

But  micrometric  measurements  are  of  particular  worth  when 
the  results  of  the  same  observer  are  compared  for  the  purpose 
of  obtaining  their  relative  value.  But  as  to  getting  the  absolute 
results  of  two  different  observers,  "one  may  confidently  maintain 
that  if  the  measurements  of  different  observers  in  any  one  in- 
vestigation are  comparable,  this  comparability  still  continues  if 
the  single  measure  selected  were  5  or  10  per  cent  greater  or 
smaller.'*5 

\Ve  have  still  some  words  to  add  concerning  the  designation 
of  the  value  which  we  obtain  by  the  micrometer.  In  former 
times  the  line  was  the  unit  of  micrometric  measurement;  in 
France  the  Paris  line  ;  in  England  the  English  duodecimal  line  ; 
in  Germany  the  Paris,  Bheinish,  or  the  Vienna  line.  Since  the 
middle  of  the  present  century  all  these  units  of  measure  have 
been  displaced  by  the  millimeter  ;  only  the  English  are  so  con- 
servative that  they  still  [partially]  maintain  the  line  unit,  just 
as  in  measuring  heat  they  will  not  exchange  the  irrational 
Fahrenheit  thermometer  for  that  of  Celsius. 

On  the  continent  the  decimal   metric  system  alone  is  used. 

6  Nageli  xind  Scbwendener,  Das  Mikroskop,  1877,  p.  285, 


130  THE  MICROSCOPE   IN  BOTANY. 

The  fractional  part  of  a  millimeter  may  be  expressed  in  two 
ways,  by  a  vulgar,  and  by  a  decimal  fraction.  Hugo  v.  Mohl6 
earnestly  pleads  for  the  use  of  the  common  fractions.  The  des- 
ignation of  micrometric  values  by  the  decimal  fraction,  he  holds 
to  be  a  real  nuisance.  He  thinks  that  one  must  be  a  mnemonic 
•expert  to  be  able  to  form  an  intuitive  conception  of  magnitude 
expressed  by  a  decimal  fraction.  With  Nageli  and  Schwendener7 
we  are  of  another  opinion.  We  believe  the  decimal  fractions 
to  be  the  only  logical  terms  in  which  to  express  micrometric 
values.  [Both  the  advantage  and  the  difficulty  of  the  decimal 
system  are  doubtless  equally  real;  but  the  perplexity  which 
prevents  persons  not  possessed  with  a  mathematical  turn  of 
.mmd  from  intuitively  apprehending  the  value  of  fractions  ex- 
..tending  to  several  places,  especially  in  decimals,  does  not  apply 
jneasurably  to  a  single  place  of  decimals,  tenths.  Hence  the 
necessity  of  a  minute  micrometric  unit,  especially,  but  not 
.exclusively,  in  a  decimal  scheme.  The  micron  (infra)  being 
frequently  sufficient  without  fractions,  and  never  requiring  any- 
thing beyond  tenths  except  for  procedures  involving  expert- 
ness  in  mathematics,  wholly  relieves  this  difficult}',  as  soon  as  it 
becomes  familiar  to  the  mind  as  a  unit  having  a  definite  value 
of  its  own.  R.  H.  W.] 

Harting8  proposed  in  his  time  to  adopt  the  one-thousandth 
part  of  a  millimeter  (0.001  mm.)  as  a  unit  for  giving  micrometric 
values.  He  would  designate  this  magnitude  by  1  mmm.,  or 
.shorter  by  It* ;  he  named  this  unit  a  micromillimeter.  This 
proposition  of  Harting  is  doubtless  the  best  and  the  microinilli- 
meter  has  maintained  itself  to  this  day.9  If  one  reads  that  an 
•object  is  23,u  long  and  knows  that  I/*  corresponds  to  the  unit  in 
the  3d  decimal  place  he  can  easily  construct  in  his  mind  the 
decimal  fraction  of  the  millimeter  which  shall  express  it  (0.023 
mm.).  But  since  we  usually  obtain  only  the  relative  size  of 
microscopic  objects,  so  even  this  latter  mental  translation  is  not 
necessary,  for  we  learn  to  think  of  the  micromillimeter  as  a  unit, 
exactly  as  in  common  life  we  do  of  the  centimeter  or  the  mark. 

«Hugo  v.  Mohl,  1.  c.,  p.  31S/— 7Nageli  tmd  Schwendener,  I.  c.,  p. 288.— « Harting,  Mikr.  p.  506. 

9  Listing  has  also  recently  pronounced  in  favor  of  the  micromillimeter  as  the  unit  for 
micrographic  measurements.  He  desires  to  introduce  the  name  Micron  or  Micrum  for  it. 
(Carl's  Repertorium  fiir  Experimentalphysik,  Bd.  x,  18G9,  p.  5.) 


MICROMETRIC    TABLES. 


131 


In  some  exceptional  cases,  in  micrometric  measurements,  we 
still  have  to  deal  with  the  old  values, — the  fractional  parts  of  a 
line.  There  are  now,  for  instance,  scarcely  any  objective  screw- 
micrometers  made,  and  those  who  use  this  instrument  will  in 
most  cases  have  to  use  an  old  one,  which  measures  in  the  frac- 
tions of  a  line.  Likewise  those  statements  of  micrometer  values 
which  occur  in  works  of  the  first  half  of  the  present  century  are 
based  on  the  Paris  line  throughout,  so  that  in  order  to  compare 
one's  own  measurements  with  those,  it  will  be  necessary  first  of 
all  to  reduce  the  latter  to  the  equivalent  fractions  of  a  milli- 
meter. I  have  found,  by  long  use,  the  tables  which  follow  to  be 
of  great  practical  convenience  in  making  these  reductions. 

%  TABLE  I. 

COMPARISON  OF  THE  UNITS  OF  MEASURE. 


1 
MM. 

PARIS 
LINE  = 

i 

ENGLISH 
LINE  = 

1 
RHEIN 
LINE  = 

1 

VIENNA 
LINE  = 

Millimeter. 

1.0000 

2.2558 

2.1166 

2.1802 

2.1952 

Paris  Line. 

0.4433,. 

1.0000 

0.9384 

0.9964 

0.9732 

English  " 

0.4724 

1.0659 

1.0000 

1.0299 

1.0371 

Rhein      " 

0.4587 

1.0347 

0.9710 

1.0000 

1.0070 

Vienna    " 

0.4555 

1.0275 

0.9642 

0.9930 

1.0000 

TABLE  II. 
REDUCTION  OF  THE  UNITS  OF  MEASURE  TO  MTCROMILLTMETERS. 


MICROMILLIMETER 

PARIS  LINE 

ENGLISH  LINE 

RHEIN  LINE 

VIENNA  LINE 

1  M  (0.001  mm.)  = 

0.000443 

0.000472 

0.000459 

0.000455 

2  "  =  0.002  mm.  = 

0.000887 

0.000945 

0.000917 

0.000911 

3  "=0.003    "      = 

0.001330 

0.001417 

0.001376 

0.001366 

4"=0.004    "      = 

0.001773 

0.001890 

0.001835 

0.001822 

5  "  =  0.005    "      = 

0.002216 

0.002362 

0.002293 

0.00-2277 

6  "  =  0.006    "      = 

0.002660 

0.002834 

0.002752 

0.002733 

7  "  =  0.007    "      = 

0.003103 

0.003307 

0.003211 

0.003188 

8"  =  0.008    "      = 

0.003546 

0.003779 

0.003670 

0.003044 

9"  =  0.009    "      = 

0.003990 

0.004252 

0.004128 

0.004099 

10  "  =  0.010     "      = 

0.004433 

0.004724 

0.004587 

0.004555 

20  "  =  0.020    "      = 

0.008866 

0.009448 

0.009174 

0.009110 

50  "  =  0.050    "      = 

0.022165 

0.023620 

0.022935 

0.022775 

1UO"  =  0.100     "      = 

0.014330 

0.047240 

0.045870 

0.045.550 

132  THE  MICROSCOPE  IN  BOTANY. 

Table  I,  "Comparison  of  the  Units  of  Measure,  "  is  in  re- 
spect to  its  purpose  perfect  enough,  but  the  calculations  involved 
in  its  use  are  somewhat  detailed.  There  will  be  necessary  in 
each  reduction  a  multiplication  at  least,  and  commonly  a  multi- 
plication and  a  division,  whereby,  on  account  of  the  many  dec- 
imal places,  an  error  might  easily  creep  in.  By  the  use  of  the 
logarithm  tables  an  addition  and  subtraction  might  be  substi- 

0  o 

tuted,  but  the  detailed  character  of  the  operation  would  not  be 
altered  materially. 

EXAMPLE  :   We  find  in  a  work  the  statement  that  a  micros- 
copic object   measures  0.0216  Paris  line.     We  wish  to  know 
how  much  this  is  in  fractions  of  a  millimeter. 
1   :  2.2558    :   :  0.0216   :  x 
x  —  0.048725  mm.  =  49,* 

Table  II,  "Reduction  of  the  Units  of  Measure  toMicromilli- 
meters,"  simplifies  the  calculations  quite  essentially.  In  it  is 
represented  the  value  of  the  different  lines  to  1-10,  20,  50,  100^ 

An  example  will  make  its  use  easily  understood  : 

1  have  found  by  my  micrometer  that  the  length  of  an  object 
is  28  ft.     In  an  old  botanical  work  I  find  the  same  object  given 
as  the  0.011841  of  a   Paris  line.     I  would  compare  these  two 
values.     I  find  the  value  of  the  28  /j.  to  be  in  fractional  part  of 
the  Paris  line,  accoiding  to  column  1. 

20  ti  —  0.008866 
8  AI  =  0.003546 

28  IL  —  0.012412 

Subtracting  the  magnitude          0.011841 

I  get  as  the  difference  0.000571     of    a   Paris    line, 

which  I  find,  by  referring  to  column  1,  is  really  a  difference  of 
something  more  than  1  //. 

EXAMPLE  2.  In  an  old  work  I  find  it  stated  that  the  cells 
of  Nephrocytium  Agardhianum  var.  minus,10  to  be  —  Paris  line. 
I  desire  to  know  how  great  this  value  is  in  terms  of  the  frac- 
tions of  a  millimeter.  ±  —  0.005000  Paris  line.  The  value 
nearest  to  that  in  my  table,  column  1,  is  0.004433  =  10  P-.  The 
difference  0.000567  lies  between  the  values  of  1  /j.  and  2  /*  near- 
est to  1  n  as  the  table  shows  it,  so  that  i  Paris  line  =  11  /*  = 
0.011  mm. 

10  C.  Nageli,  Gattungen  einzelliger  Algen,  Zliricb,  1849,  p.  80. 


POLARIZING  APPARATUS  AND  GONIOMETER.  133 

Calculations  for  the  English  and  other  lines  are  made  in*  the 
same  way,  using  of  course  the  corresponding  columns  in  the 
table.  We  have  not  considered  it  necessary  to  give  examples 
for  each  kind  of  line. 

[The  systems  in  use  in  micrometry  in  this  country,  and  in 
England,  are  the  English  and  the  metric.  Of  the  English  inch, 
vulgar  fractions  are  more  commonly  used  than  decimals  ;  partly, 
perhaps,  because  so  many  of  those  persons,  whose  education  and 
habits  would  lead  them  to  choose  decimals,  are  led  to  adopt  the 
consistent  decimal  or  metric  system,  to  the  exclusion  of  the 
inch.  The  use  of  the  line  seems  to  have  been  abandoned.  It 
can  scarcely  be  doubted  that  there  is  a  growing  disposition  to 
use  the  metric  units  in  microscopy,  the  millimeter  (approxi- 
mately ^  inch)  for  a  large  unit,  and  the  micromillimeter  (approxi- 
mately JL  inch)  for  a  small  unit;  and  persons  who  have  once 
learned  to  think  in  these  units,  so  as  to  avoid  the  trouble  of 
constant  computation,  find  them  most  convenient  and  satisfac- 
tory in  themselves,  to  say  nothing  of  the  manifest  advantages 
of  harmony  with  the  rest  of  the  world.  The  table  opposite,  from 
the  Journal  of  the  Royal  Microscopical  Society,  presents  in 
n  most  available  form  the  relations  to  each  other  of  the  various 
English  and  metric  units  and  their  fractions.  By  following, 
with  this  table,  the  directions  of  the  author  (p.  132)  for  the  use 
of  his  w  Table  II,"  observations  in  inches  and  those  in  millime- 
ters or  micromillimeters  can  be  transposed  with  great  facility. 
Moreover  the  table  furnishes  the  necessary  information  in  regard 
to  metric  measures  of  length,  to  any  who  may  be  as  yet  familiar 
with  only  the  English  system.  R.  H.  W.] 

IV.     POLARIZING   APPARATUS  AND  GONIOMETER. 

It  does  not  come  within  the  plan  of  this  work  to  consider 
critically  the  process  of  the  polarization  of  light.  Whoever 
wishes  to  go  into  the  subject  may  consult  any  modern  work  on 
physics.  For  microscopical  purposes  the  subject  is  developed 
in  the  best  form  by  Nageli  and  Schwendener  in  "  The  Micro- 
scope,"11 and  in  the  work  of  like  name  by  Dippel,12  though  the 

11 II  Auflagc  (1877)  pp.  239-361.  "  Bel.  I,  p.  224-227,  pp.  407-455. 


134  THE  MICROSCOPE  IN  BOTANY. 

presentation  of  the  matter  by  the  latter  is  far  less  satisfactory 
to  me  than  that  by  the  former. 

The  microscopical  polarizing-apparatus,  like  every  other, 
comprises  an  arrangement  of  two  polarizing  media  between 
which,  in  the  polarized  light,  the  object  to  be  investigated 
may  be  examined.  The  one  placed  under  the  object  is  called 
the  polarizer  and  the  one  over  it  the  analyzer.  These  condi- 
tions in  general  characterize  the  positions  of  the  polarizing  ap- 
paratus of  the  microscope.  The  polarizer  is  most  conveniently 
arranged  in  connection  with  the  stage.  Since  it  is  of  a  cylin- 
drical form  as  at  present  made  it  may  be  put  into  the  place  of 
the  cylinder  diaphragm.  Naturally,  the  analyzer  cannot  go  be- 
tween the  objective  and  the  object,  and  it  is  therefore  placed 
either  directly  over  the  objective-system  or  above  the  ocular. 
All  [Continental]  makers  now  follow  Hartnack  in  adopting  the 
latter  arrangement.  As  a  polarizing  medium,  the  Nicol's  prism 
of  Iceland  spar  is  the  one  universally  adopted. 

As  is  well  known  a  Nicol's  prism  consists  of  two  halves ; 
the  joining  surfaces  run  diagonally  through  the  prism  and  form 
an  angle  with  the  end  surfaces  of  89°  17'.  The  two  halves  are 

o 

cemented  together  with  Canada  balsam.  The  side  surfaces 
are  blackened.  If  a  ray  of  light  running  parallel  to  the  side 
surfaces  falls  upon  the  lower  end  of  the  prism  it  is  separated 
into  two  rays,  the  ordinary  and  the  extraordinary.  The  former 
suffers  a  total  reflection  in  the  layer  of  Canada  balsam  and 
passing  to  the  side  of  the  prism  is  absorbed  in  the  blackened 
surface ;  the  latter,  on  the  contrary,  passes  directly  through  the 
prism  into  the  field  of  view  of  the  microscope  which  it  illumin- 
ates. 

It  is  known  that  in  the  use  of  the  polarizing  apparatus  the 
mutual  position  of  the  polarizing  media  must  be  changed,  and 
it  is  all  the  same  if  the  polarizer  be  turned  about  while  the 
analyzer  remains  fixed,  or  the  analyzer  be  rotated  while  the 
polarizer  is  fast.  One  or  both  must  be  rotated  around  its  longer 
axis  —  the  axis  parallel  with  its  sides. 

The  Nicol's  prisms,  the  side  edges  of  which  belong  to  the 
original  rhomboidal  calc  spar,  have  a  vertical  direction,  while 
the  artificially  ground  edges  exposed  above  and  below  are  set 


POLAKIZIXG  APPARATUS.  135 

at  an  angle  of  68°.  [They  are  set,  by  means  of  a  soft  cork 
packing,  in  brass  tubes  adapted  to  the  instrument  for  which 
they  are  designed,  and  the  exposed  ends  are  by  some  makers 
protected  by  thin  cover-glasses.  The  polarizer,  as  mounted, 
is  slipped  into  the  substage  ring,  and  in  the  better  class  of  in- 
struments can  be  used  in  connection  with  and  not  in  the  place  of 
the  substage  condenser.  It  nearly  always  has  a  rotating  move- 
ment, and  in  stands  designed  for  chemical  or  lithological  work 
a  graduated  arc  by  which  its  position  can  be  known  and  re- 
corded. It  is  advantageous  to  have  this  prism  as  large  as  is 
consistent  with  the  construction  of  the  stand.  The  analyzing 
prism  is  mounted  in  a  smaller  tube.  It  may  be  a  rather  small 
prism  and  should  be  short.  It  is  sometimes  mounted  in  a  cap 
to  slide  over  the  top  of  the  ocular,  as  near  as  possible  to  the 
eye  lens,  but  is  more  commonly  fitted  as  in  Fig.  55  into  an 


FIG.  55. 

adapter  with  society  screw  above  and  below,  to  be  screwed  in 
between  the  objective  and  the  tube  of  the  microscope.  It  is 
frequently  so  arranged,  by  cutting  away  the  sides  of  the  con- 
taining adapter,  that  it  can  be  easily  rotated  when  in  use.  By 
the  addition  of  a  light  brass  tube,  just  large  enough  to  contain 
this  apparatus  above  and  to  slip  over  the  cap  tube  of  the  ocu- 
lar below,  the  writer  has  been  able,  with  great  satisfaction,  to 
secure  the  advantages  of  both  methods  of  locating  the  analyzer, 
which  can  thus  be  placed  at  will  in  either  position.  Fig.  55 
shows  us  a  common  method  of  mounting  the  polarizing  and 
analyzing  prisms  respectively.  R.  H.  W.] 


136  THE  MICROSCOPE  IN  BOTANY. 

Use  of  the  polarizing  apparatus.  As  is  well  known  the  po- 
larizing apparatus  is  commonly  employed  to  find  out  if  a  given 
object  —  for  example  a  crystal  —  has  one  or  two  optical  axes, 
if  it  be  singly  or  doubly  refractive.  If  the  crystal  be  of  mi- 
croscopical minuteness  one  naturally  uses  a  microscopical  polar- 
izing-apparatus.  On  this  account  it  plays  an  important  role  in 
the  hands  of  the  crystallographer.  But  it  is  frequently  of  the 
greatest  use  to  the  botanist.  For,  in  the  first  place  it  enables 
him  to  know  the  nature  of  many  of  the  crystals  which  occur 
within  the  plant-cells,  and  in  the  second  place  all  tissue  struc- 
tures are  doubly  refractive  and  may  be  examined  with  polar- 
ized light.  Not  seldom,  details  of  structure  show  themselves 
when  they  are  illuminated  thus  in  the  dark  field  of  view  which 
are  otherwise  not  seen  at  all,  or  recognized  with  the  greatest 
difficulty,  and  thirdly  the  polarizing  apparatus  enables  the  bot- 
anist clearly  to  make  out  the  form  of  certain  microscopic 
objects. 

For  an  experimental  observation  with  the  polarizing  appara- 
tus, starch  grains  from  a  potato,  or  a  section  of  the  rhizome  of 
Pteris  aquilina,  in  which  the  ducts  of  the  fibro-vascular  bundles 
alone  are  doubly  refractive,  will  serve  us  excellently  well  for  an 
object.  A  very  good  object  also  is  a  section  of  the  under- 
ground stem  of  Lathraea  squamaria  for  in  this  we  have  the 
starch  grains  and  the  doubly  refracting  fibers  together. 

In  the  use  of  the  apparatus  we  proceed  as  follows.  After 
we  have  adjusted  the  object  by  means  of  a  common  ocular  and 
found  the  best  illumination  by  turning  the  mirror  about,  we 
place  the  polarizer  in  position  beneath  the  stage.  The  field 
appears  to  be  unchanged,  except  a  slightly  weaker  illumination. 
After  placing  the  analyzer  also  in  position,  whether  above  the 
ocular  or  .above  the  objective,  we  rotate  the  movable  part  of 
the  apparatus  till  we  bring  the  two  prisms  into  such  a  mutual 
position  that  the  field  of  vision  appears  darkest.  If  the  appar- 
atus is  well  constructed  the  darkening  is  almost  total.  The 
field  appears  in  a  quite  deep,  very  agreeable,  shade  of  blue. 
With  the  use  of  low  powers  there  is  a  certain  amount  of  light 
falling  upon  the  object  from  the  side  which  is  reflected  up- 
wards, and  the  consequence  is  that  the  field  is  not  perfectly 


THE  GONIOMETER.  137 

dark.  This  difficulty  can  be  remedied  by  holding  the  hand  so 
as  to  shade  the  object  on  the  stage,  or  putting  round  about  it 
for  a  shade,  a  piece  of  angularly  folded  common  blue  wrapping 
paper.  The  doubly  refracting  parts  of  an  object  are  seen  on 
a  dark  ground,  either  partly  or  altogether  brightly  illuminated, 
according  to  their  chemical  or  physical  structure.  Thus 
starch  grains  from  the  potato  appear  of  a  clear  bluish  shade  with 
a  dark  cross  drawn  over  them  whose  radii  broaden  outwardly 
toward  the  circumference.13  The  walls  of  the  ducts  from  the 
rhizome  of  the  Pteris  appear  illuminated  partly  bluish  and 
partly  yeliowish.1*  As  already  indicated,  we  cannot  here  in 
the  least  enter  into  a  discussion  of  the  optical  characteristics 
of  organic  forms,  but  must  rather  recommend  the  careful  study 
of  those  parts  of  Nageli  and  Schwendener's  as  well  as  of  Dip- 
pel's  work,  which  deal  with  this  subject. 

Rotating  selenite  plates,  which,  as  is  well  known,  are  applied 
in  various  ways  to  the  common  polarizing  apparatus,  can  be 
easily  added  also  to  the  microscopical  polarizing-apparatus. 

[THE  GONIOMETER.] 

[For  the  purpose  of  measuring  the  angles  of  microscopic 
objects,  the  goniometer  is  frequently  added  to  the  microscope. 
This  attachment  originally  consisted  of  an  elaborately  mounted 
ocular  in  whose  field  of  view  was  a  line  which,  by  rotating  the  ocu- 
lar, could  be  brought  successively  into  coincidence  with  the  dif- 
ferent sides  of  the  object,  the  intervening  angles,  traversed  by  the 
rotating  apparatus  being  read  off  by  means  of  an  attached  vernier 
sliding  over  a  graduated  circle  clamped  to  the  microscope  tube. 
This  accessory  may  well  be  combined  with  thepolariscope,  as  it 
is  frequently  used  in  connection  writh  studies  which  require 
polarized  light.  Such  micro-goniometers  are  still  attached  to 
stands  designed  for  chemical  or  lithological  work,  but  the  in- 
troduction of  the  circular  concentric  stage  has  rendered  them 
unnecessary  except  for  the  use  of  the  specialists.  The  stand 
figured  in  Plate  XI,  for  instance,  can  be  employed  for  gouiom- 

13  The  reasons  therefor  may  be  found  in  the  before  cited  works. 

14  For  this  matter  see  principally  Dippel,  L  c. 


138  THE  MICKOSCOPE  IN  BOTANY. 

etry  without  addition  or  preparation  ;  and  any  of  the  circular- 
stage  stands  will  require  for  the  same  purpose  only  a  graduated 
circle  on  the  edge  of  the  stage.  As  such  graduation  is  inex- 
pensive, and  is  also  useful  for  other  purposes,  it  constitutes, 
for  occasional  and  incidental  use,  the  most  eligible  goniometer. 
For  the  measurement  of  angles  the  stage  must  be  carefully 
centered,  and  the  angle  to  be  measured  brought  to  the  centre  of 
the  field  of  view.  Such  adjustment  is  facilitated  by  placing 
cross  lines  at  the  focus  of  the  eye-lens  of  the  ocular  as  shown 
in  Fig.  56,  but  only  one  line  fc  is  essential.  This  line  may 

be  either  a  spider's  thread  drawn 
across  the  center  of  the  opening  of 
the  diaphragm  of  the  ocular,  or  a 
line  ruled  on  a  thin  cover  glass  lying 
in  the  same,position,  one  of  the  lines 
of  an  ocular  micrometer  being  often 
made  to  answer  the  purpose.  An 
angle  whose  plane  is  parallel  to  the 
plane  of  the  stage,  and  at  right 
angles  to  the  axis  of  vision,  is 
carefully  selected  and  its  apex 

brought  to  the  center  of  the  field.  The  line  in  the  ocular 
is  then  made  to  coincide  with  one  of  its  sides,  and  the  stage  is 
afterward  rotated  until  the  other  side  coincides  with  the  same 
line,  the  angle  through  which  the  stage  and  object  have  moved 
being  readily  determined  by  comparing  readings  from  the  stage 
graduation  made  before  and  after  the  rotation.  Should  greater 
precision  be  required  a  vernier  may  be  added  to  give  decimals 
of  a  degree ;  but  this  is  not  always  needed,  all  measurements 
with  any  micro-goniometer  being  at  best  but  approximate.  It 
is  seldom  if  ever  possible  to  be  certain  of  the  exact  parallelism 
of  a  given  plane  of  the  object  with  that  of  the  stage,  and  un- 
less that  parallelism  be  secured  it  is  evident  that  the  angle  will 
be  seen  in  perspective  and  will  be  incorrectly  stated  in  the  read- 
ing of  the  instrument.] 

[Mr.  Zentmayer  combines  a  goniometer  attachment  with  his 
cobweb  micrometer  as  shown  in  Fig.  54,  the  lower  graduated 
circle  being  firmly  attached  to  the  microscope  body,  and  the  ver- 


THE  MICRO-SPECTROSCOPE. 


139 


nier  above  it  enabling  us  to  read  off  accurately  the  extent  to  which 
the  micrometer  with  its  field  crossed  by  fine  lines  has  been  ro- 
tated. A  pair  of  extra  lines,  crossing  each  other  in  the  center, 
are  provided  for  the  accurate  centering  of  the  angle  to  be 
measured.  The  analyzing  prism,  when  required  with  this  com- 
bination, is  placed  above  the  objective.  R.  H.  W.] 

Y.    TPIE  MICRO-SPECTROSCOPE. 

In  recent  times  investigations  are  frequently  carried  on  by 
means,  of  a  spectroscopic  apparatus  combined  with  the  micro- 
scope. The  spectro-microscopical  apparatus,  especially  in  the 
hands  of  botanists,  has  become  an  important  instrument  in 
the  investigation  of  the  coloring  matter  of  plants.  Since  we 
have  found  an  adequate. description  of  the  micro-spectroscopic 
apparatus15  in  no  existing 
work,  we  shall  here  attempt 
to  consider  it  somewhat  in 
detail.  We  shall  found  our 
description  on  that  most 
perfect  construction  of  it 
which  was  first  given  to  it 
by  Sorby  and  Browning. 

A.  The  Prisms.  It  is 
well  known  that  when  a  ray 
of  so-called  white  light  pass- 
es  through  a  massive  glass 
prism,  provided  that  the  re- 
fracting edge  of  the  prism  be 
perpendicular  to  fhe  ray  of 
light,  it  will  be  separated 
into  its  elementary  colors. 
Then  there  is  produced  a 
spectrum  (solar  spectrum) 
whose  colors  and  their  arrangement  are  well  known.  Now,  if  in 
place  of  the  dispersing  prism  we  substitute  a  combination  of 
three  or  five  prisms  of  crown  and  flint  glass  alternately, 
arranged  as  is  shown  in  Fig.  57  at  A9  the  light  will  pass  through 

«  Sageli  and  Schwendener,  L  c.t  p.  39.— Frey,  Das  Mikroskop,  p.  36, /. 


FIG.  57. 


140 


THE  MICROSCOPE  IN  BOTANY. 


it  in  a  direction   parallel  to   its   axis.      This    arrangement  is 
here  known  as  a  direct16  vision  spectroscope. 

The  prisms  are  fastened  into  a  brass  cylinder  M,  by  means  of 
a  cork  mounting.  The  cylinder  is  closed  above  by  a  dull  black 
metal  plate  K,  with  a  round  hole  in  the  middle  about  10  mm. 
wide  for  looking  into.  This  apparatus  is  connected  with  the 
ocular  and  is  placed  above  the  eye-lens  G.  Should  the  ocular 
have  a  common  circular  diaphragm,  the  field  of  the  microscope, 
after  putting  the  spectroscopic  apparatus  in  place,  should  appear 
to  be  a  small  ellipse,  the  center  of  which  should  be  quite  colorless, 
and  at  the  points  of  greatest  curvature  red  and  blue  respectively. 
Microscopic  objects  which  do  not  fill  the  field  seem  to  be  striated 
in  the  direction  of  the  longer  diameter  of  the  ellipse.  If  in 

place  of  the  diaphragm-opening 
of  the  spectroscopic  ocular  we 
substitute  a  slit-opening  which 
is  so  arranged  in  respect  to 
the  prisms  that  the  refracting 
edge  is  parallel  to  the  slit  B, 
a  spectrum  will  now  appear 
of  the  well-known  band-form 
whose  brightness  and  extension 
are  conditioned  by  the  breadth 
and  length  of  the  slit. 

B.  The  Slit.  To  constitute 
a  practical  working  instrument 
the  slit  must  be  exactly  adjusta- 
ble in  the  focus  of  the  eye-lens 
of  the  ocular,  for  every  eye, 
and  it  must  be  so  contrived 
that  the  slit  may  be  narrowed  or  widened,  lengthened  or 
shortened  at  will. 

The  focussing  of  the  slit  is  easily  provided  for  by  having  the 
tube  of  the  ocular  made  of  two  parts,  one  shoving  into  the 
other  and  moved  by  a  rack  and  pinion. 

[The  contrivances  for  widening  the  slit  are  about  as  various 

is  This  combination  was  first  contrived  by  Amici  in  1803,  later  applied  to  the  construction 
of  a  pocket  spectroscope  by  Hofman  (see  Schellen,  Spectralanalyse,  p.  109)  and  finally 
proposed  for  the  microscope  by  John  Browning. 


FIG.  58. 


THE  COMPARISON  PRISM. 


141 


as  the  ideas  of  the  different  makers.  It  is  only  essential  that 
the  two  shutters  forming  the  sides  of  the  slit  should  steadily 
approach  and  finally  meet  each  other  in  the  center  of  the  field, 
with  a  perfectly  smooth  and  parallel  motion,  which  is  under  com- 
plete and  easy  control.  In  Fig.  57  this  motion  is  controlled  by 
a  small  milled  head.  Shutters  moved  by  the  lever  L  are  pro- 
vided for  controlling  the  length  of  the  slit.  R.  H.  W.] 

C.  The  Comparison  Prism.  It  is  often  very  desirable  to 
compare  the  spectrum  of  a  body  which  is  being  investigated 
with  that  of  a  like  body.  This  may  be  done  by  first  observing 
the  spectrum  of  the  body  under  investigation,  and  after  that 
the  spectrum  of  the  body  used  for  comparison.  But  the  spec- 


FIG.  59. 

tra,  for  example  the  absorption  spectra  of  some  colored  fluids 
with  many  dark  bands,  are  found  to  be  very  difficult  to  carry 
in  the  memory  so  as  to  make  the  comparison.  Hence  a  com- 
parison of  the  spectra  in  this  way  can  relate  only  to  the  leading 
features  and  not  to  smaller  particulars.  But  in  order  to  make 
comparison  of  the  finer  details  with  sufficient  leisure  and  exact- 
ness we  must  see  both  spectra  very  near  together,  and  at  the 
same  time  —  the  so-called  double  spectrum.  This  we  can  do 
by  means  of  the  comparison  prism,  an  apparatus  for  which  we 
are  indebted  to  Kirchhofl'.17 

Suppose  tl,  Fig.  59, 1,  be  the  plate  of  the  drum  which  bears  the 

17  Schelling,  Spectrum  analysis  in  its  application  to  the  substance  of  the  earth  and  the 
nature  oi  the  heavenly  bodies,  Brunswick,  1870,  p.  121. 


142 


THE  MICROSCOPE  IN  BOTANY. 


slit  s.  The  body  to  be  investigated  is  at  b.  The  rays  from  it 
pass  in  the  direction  lib'  through  the  slit  and  at  b'  enter  the 
combination  of  refracting  prisms.  The  slit  is  not  opened  along 
its  whole  length  for  the  admission  of  rays  from  b.  For  half 
of  its  length  it  is  covered  by  a  reflecting  prism  of  the  form 
which  we  have  come  to  know  in  the  apparatus  for  microscopical 
drawing  (see  p.  114,  Fig.  45).  The  prism  in  section  represents 
a  right  angled  isosceles  triangle,  Fig.  59,  II,  the  hypothenuse 
surface  being  inclined  to  the  slit  at  an  angle  of  45°.  Now,  if 
we  suppose  another  body  to  be  used  for  comparison  placed  so 
as  to  send  rays  from  a  in  a  horizontal  direction,  they  fall  upon 
the  surface  of  the  prism  perpendicular^,  and  unrefracted  pass 
to  the  hypothenuse  surface  and  fall  upon  it  at  an  angle  of  45°. 
Here  they  are  totally  refracted  perpendicularly  upwards,  and  as 
a'  pass  through  the  slit  parallel  with  the  rays  bb'.  If  now  the 
refracting  prisms  are  placed  over  the  slit  as  already  described 
the  observer  will  now  perceive  not  one  but  two  spectra  lying 
next  each  other  whose  colors,  Fraunhofer  lines,  etc.,  exactly  coin- 
cide. We  call  these  two  a  double  spectrum. 


FIG.  GO. 


Fig.  60  represents  a  double  spectrum  which  is  produced  by 
the  rays  coming  through  the  free  half  of  the  slit,  and  by  those 
of  diffused  daylight  passing  through  the  comparison  prism.  I 
is  the  spectrum  of  the  rays  through  the  prism,  II  that  of  those 
not  reflected.  They  are  separated  by  a  slender  black  line  which 
one  commonly  notices  but  which  when  one  has  the  eye  close 
over  the  refracting  prisrn  has  no  disturbing  influence.  We 
notice  in  the  spectra  the  strong  Fraunhofer  lines :  those  of  the 
under  spectrum  falling  exactly  in  the  prolongation  of  those  of 
the  upper.  [The  comparison  prism,  not  being  required  for 


THE  BINOCULAR  MICRO-SPECTROSCOPE. 


143 


FIG.  61. 


constant  use,  is  mounted  in  a  sliding  frame  and  can  be  instantly 
slipped  into  or  out  of  the  field  of  view  by  means  of  the  milled 
head  I,  Fig.  57.  K.  H.  W.] 

Laterally,  on  the  largest  tube  or  drum,  is  a  perpendicularly  ar- 
ranged plate  E,  Fig.  57,  provided  with  spring  clamps,  which  has 
the  form  of  a  microscope  stage  before  which  may  A 

be  seen  a  plane  mirror  F.  The  plate  has  a  small 
opening  in  the  middle  at  (7which  shouldbe  closed 
when  the  comparison  plate  is  not  in  use.  If  one 
wishes  to  use  the  comparison  prism  he  pushes 
the  milled  head  I  forward  and  the  prism 
which  has  been  lying  in  the  side  of  the  drum 
is  shoved  into  place  over  one-half  of  the  slit. 
By  proper  adjustment  of  the  mirror  the  neces- 
sary light  can  be  directed  through  the  open- 
ing C  in  the  stage  E  upon  the  comparison 
prism.  For  the  production  of  the  comparison 
spectrum  we  should  bring  the  comparing  body 
as  closely  as  possible  before  opening  in  the  plate  E. 

[/>.  The  Binocular  Micro- Spectroscope.  The  spectroscopic 
ocular  hitherto  mentioned  is  the  original  form  contrived  by  Mr. 
H.  C.  Sorby  of  London  and  elaborated  by  John 
Browning  a  distinguished  manufacturing  opti- 
cian of  that  city.  It  is  still  the  form  in  most 
general  use.  It  is  shown  in  Fig.  58  complete  and 
ready  to  be  inserted  in  any  micro  -cope  in  place  of 
the  usual  ocular.  Mr.  Sorby  has  more  recently 
contrived  another  form  known  as  the  Binocular 
spectroscope.  In  this  arrangement,  Fig.  62, 
and  shown  in  section  in  Fig.  61,  the  prisms  are 
transferred  from  above  the  ocular  to  below  the 
H  objective,  giving  greater  dispersion  as  well  as 
fitness  for  binocular  use.  The  apparatus  screws 
into  the  tube  of  the  microscope,  in  place  of  the 
objective  by  the  screw  B.  It  consists  of  a  small  collecting  lens 
/  which  when  in  use  should  just  touch  the  object  under  investi- 
gation ;  a  slit  adjustable  by  the  screw  head  L  and  a  set  of  prisms 
c  through  which  light  from  /passes  upward  to  the  objective  A 


FIG.  62. 


144 


THE  MICROSCOPE  IN  BOTANY. 


and  thence  through  the  microscope  tube  and  ocular.  The  com- 
parison prism  G  can  be  made  to  send  up  either  the  absorption 
bands  of  a  known  solution  or  the  interference  spectrum  from 
the  standard  scale  H.  This  apparatus,  like  the  preceding,  is  of 
the  Beck  style,  supplied  in  this  country  by  Wm.  H.  Walmsley 
and  Co.  of  Philadelphia.  K.  H.  W.] 

E.  The  Measuring  Apparatus  of  the  Micro-spectroscope \ 
It  is  known  that  Bunsen  and  Kirchhoff,  in  their  chemical  inves- 
tigations upon  the  absorption  spectra  and  the  Fraunhofer  lines, 
divided  the  whole  length  of  the  spectrum  into  170  equal  parts, 
and  determined  the  position  of  the  Fraunhofer  lines  in  the  solar 


m 


FIG.  G3. 


spectrum  and  the  illuminated  bands  in  the  discontinuous  spec- 
trum according  to  a  scale.  In  general  this  scale  is  fundamental 
in  spectrum  analysis.  The  common  spectroscope  for  the  pur- 
pose of  spectrum  analysis  has  a  tube,  in  the  front  end  of  which 
is  a  glass  on  which  is  photographed  a  millimeter  scale  reduced 
about  fifteen  times.  It  can  be  illuminated  by  light  admitted 
for  the  purpose.  The  image  of  this  scale  is  thrown  by  means 
of  a  biconvex  lens  on  the  front  surface  of  the  refracting  prism, 
this  is  reflected  into  the  observing  telescope  and  reaches  the  eye 
of  the  observer  as  an  optical  image  apparently  lying  on  the 


THE  MICRO-SPECTROSCOPE.  145 

spectrum.  The  contrivance,  however,  suffers  from  this  fault 
that  the  thickness  and  brightness  of  the  division  lines  are  de- 
pendent on  the  width  of  the  slit,  which  is  different  with  differ- 
ent eyes.  But  when  we  undertake  to  measure  the  exact  distance 
of  the  Fraunhofer  lines  and  the  breadth  of  the  absorption  bands 
we  have  to  give  up  this  very  simple  and  practical  contrivance. 
The  idea  of  an  exact  measuring  apparatus  for  the  micro-spec- 
troscope, which  we  may  call  really  ingenious,  originated  with 
Mr.  John  Browning  of  London.  This  not  very  simple  contriv- 
ance is  illustrated  in  section  in  Fig.  63.  First  is  rr  the  per- 
pendicular brass  tube  which  incloses  the  five  prisms  ww,  in  their 
cork  mounting  xx.  The  upper  surface  of  the  prism  w  Y  is  inclined 
to  the  horizon  at  an  angle  of  45°.  Opposite  to  it  is  the  hori- 
zontal tube  ran,  provided  within  with  a  biconvex  lens  <?,  which  is 
movable  in  the  direction  of  the  length  of  the  tube  tin.  This 
tube  nn  bears  upon  its  front  fastened  to  it  the  brass  plate  ee. 


EC 

«       -_  ; 

"      N     1     .      |            -I 

FIG.  64. 

In  a  dovetail  guide  on  this  (movable  longitudinally)  is  the 
plate  g  on  which  a  narrow  brass  tube  bb  is  securely  fastened. 
The  latter  is  closed  at  c  with  a  tinfoil  plate  in  the  middle  of 
which  is  made  a  very  small  opening  of  the  form  of  an  equilat- 
eral triangle.  Outside,  bb  carries  a  double  arm  on  which  the 
small,  movable,  plane  mirror  a  is  held.  Now,  by  illuminating 
the  triangle  at  c,  by  means  of  the  mirror  a  and  giving  the  lens 
q  the  right  adjustment  for  the  eye  of  the  observer,  we  shall  see, 
by  bringing  the  eye  to  the  point  <5,  a  white  illuminated,  magni- 
fied image  of  the  triangle.  The  course  of  the  rays  and  their 
double  reflection  are  indicated  by  the  dotted  line  afrd.  If,  now 
we  suffer  the  light  to  pass  through  the  prisms  ww,  we  shall  per- 
ceive a  spectrum  on  which  the  triangle  seems  to  be  lying.  By 
a  suitable  adjustment  of  the  whole  apparatus  it  can  be  brought 
about  so  that  the  image  of  the  triangle  will  appear  within  the 
spectrum  or  upon  its  upper  or  lower  border.  Fig.  64,  for 
10 


146 


THE  MICROSCOPE  IN  BOTANY. 


example,  shows  a  single  solar  spectrum  on  the  upper  edge  of 
which  this  measuring  triangle  lies. 

In  order  that  this  ingenious  contrivance  may  be  useful  in 
measuring  distances  on  the  spectrum,  the  triangle  must  be 
movable  along  the  length  of  the  spectrum  and  the  amount  of 
the  movement  must  be  capable  of  the  most  exact  measurement. 
We  must  keep  in  mind,  however,  that  all  determinations  of 
length  in  the  spectrum  represent  only  relative  size.  There  is  no 
such  thing  here  as  determining  absolute  magnitudes. 


FIG.  65. 

The  contrivance  which  serves  our  purpose  in  this  is  quite  the 
same  as  the  one  already  described  in  the  objective  screw-micro- 
meter. It  is  diagrammatically  sketched  in  Fig.  63.  The  plate  ee 
carries  in  a  brass  projection  gf  a  micrometer-screw  Jc  which  ro- 
tates but  does  not  progress.  The  thread  of  the  screw  works  in 
a  like  projection  of  the  plate  which  carries  the  triangle  c.  By 
turning  the  screw  this  is  moved  up  or  down  in  a  dovetailed 
bearing  or  guide  on  ee.  The  amount  of  this  movement  which 

o  o 

corresponds  to  the  size  of  the  screw  thread  may  be  read  oif  on 
rulings  engraved  upon  the  inner  plate  and  playing  by  an  index 
on  the  outer ;  fractions  of  the  same  may  be  read  from  the  drum 
ii  whose  periphery  is  divided  into  100  parts. 

We  shall  now  understand  the  illustration,  Fig.  65,  which  rep- 
resents the  whole  measuring  apparatus  in  about  |  its  natural 
size.  The  letters  a  b  k  g  g'  ilm  n  u  and  r  are  of  like  signifi- 


THE  MICRO-SPECTROSCOPE.  H7 

cance  with  the  corresponding  ones  in  Fig.  63.  The  two  metal 
arms  on  which  the  mirror  is  mounted  and  rotates  are  represented 
by  a1  a'.  They  spring  from  the  brass  ring  b'  b'  which  rotates  on 
bb.  At  V  we  see  the  index  by  which  the  drum  with  its  rulings 
passes,  and  z  is  a  small  handle  by  means  of  which  the  lens  q  is 
moved  and  the  image  of  the  triangle  adjusted.  Naturally,  the 
tube  u  must  fit  very  exactly  upon  rr  which  holds  the  prisms, 
or  the  measuring  would  be  very  inaccurate. 

In  order  to  measure  by  means  of  this  contrivance,  the  spec- 
troscope is  taken  off  the  microscope  tube,  and  the  measuring 
apparatus  carefully  adjusted  and  then  replaced.  The  little  mir- 
ror on  the  transverse  tube  must  be  so  arranged  that  the  light 

O  O 

will  fall  upon  the  triangle  and  then  the  lens  should  be  adjusted 
by  means  of  the  little  knob  in  the  tube.  To  obviate  any  paral- 
Lictic  displacement  during  the  measuring,  this  adjustment  must 
be  such  that  it  and  the  Fraunhofer  lines  adjusted  by  means  of  the 
pinion  jV,  Fig.  57,  come  to  exactly  the  same  visual  distance.  We 
may  know  when  this  two-sided  adjustment  is  exactly  right  by 
the  fact  that  a  movement  of  the  head  from  side  to  side  xloes  not 
show  the  triangle,  which  has  been  placed  on  a  given  line,  mov- 
ing across  the  lines.  If  the  apparatus  is  made  with  precision 
it  will  allow  of  measurements  of  extraordinary  exactness.  We 
should  not  remove  the  apparatus  from  the  microscope  till  after 
the  measuring  is  finished  for  the  removal  and  replacing  of  the 
instrument  might  easily  make  a  difference  of  several  divisions 
of  the  drum  head  in  the  distances  determined. 

Each  observer,  before  he  can  use  the  instrument  in  investiga- 
tions, must  provide  himself  with  a  scale  (which  for  eyes  of 
different  visual  lengths  will  be  different),  in  which  the  position 
of  the  several  Fraunhofer  lines  are  exactly  designated.  This, 
once  constructed,  serves  as  the  foundation  of  all  measurements. 
It  will  commonly  be  sufficient  when  we  have  determined  the 
distance  of  the  Fraunhofer  lines  A  to  HOY  H  (see  Fig.  72,  p. 
153)  in  divisions  of  the  measuring18  apparatus  and  with  this  unit 
the  distance  apart  of  the  lines  A,  a,  B,  C,  D9  E,  F,  6r,  as 
found  by  measuring.  If  by  subsequent  measurements  one 

19  With  the  apparatus  (Seibert  ami  Kraft)  which  I  have  useJ  and  my  eye,  the  distance 
from  A  to  H  is  7 J2  graduations  of  the  drum. 


148  THE  MICROSCOPE  IN  BOTANY. 

would  eliminate  every  possible  error  he  should,  after  the  com- 
pletion of  such  a  scale,  focus  the  triangle  on  a  given  Fraunhofer 
iliue  and  then  see  if  it  lies  exactly  on  the  point  designated  in 
the  scale.  A  casual  difference  may  arise  in  the  calculation. 

Example  for  the  Application  of  the  Measuring 
Apparatus. 

We  here  addc,  concrete  example  to  illustrate  what  has  just 
been  said. 

It  is  required  to  measure  the  relative  distance  of  the  Fraun- 
hofer lines  D,  E,  b,  F. 

We  focus  the  triangle  on  the  upper  edge  of  D  and  notice  the 
position  :=«223.3.  Then  turn  the  micrometer-screw  forward  to 
E  and  read  off  =  362.5.  Distance  then  from  D  to  E  —  139.2. 
Now  turn  forward  to  b  —  382.5.  Distance  from  E  to  b  —  20. 
Finally  turn  to  F,  read  —  490.7.  Distance  from  b  to  F,—  108.2. 
According  to  a  lithograph  spectrum  table  of  Bunsen  and  Kirch- 
hoff  founded  on  the  scale  of  170  divisions  (see  p.  144)  there  is 
given  D  to  .#=21.2,  b  to  F—  15.7. 

Comparing  the  distances  D  E,  and  b  F,  according  to  our 
measurement  %£  =  %£  =  1.2865 

Comparing  the  same  distances  £jf  —  ?il  =  1.293  accordimg 
'to  Buusen's  table. 

Difference  of  the  two  values  0.0065. 

[The  Standard  Scale,  also  devised  by  Mr.  Sorby,  is  a  simple 
and  convenient  means  of  measuring  the  position  of  absorption 
bands,  when  great  precision  is  not  required.  It  consists  of  a  plate 
of  quartz  cut  parallel  to  the  principal  axis  of  the  crystal  to  the 
thickness  of  about  1.09  mm.,  and  mounted  between  two  Nicol's 
prisms  or  Herapathites.  When  light  is  transmitted  through  this 
combination,  it  is  evident,  from  the  well-known  property  of 
quartz  under  polarized  light,  that  an  interference  spectrum  will  be 
formed.  This  spectrum  is  divided  into  twelve  optically  equal 
parts  by  black  bands,  the  third  space  counting  from  the  red 
end  towards  the  blue  corresponding  exactly  to  the  position  of 
the  D  or  sodium  line.  The  optical  parts  are  enclosed  in  a 
tube  attached  to  a  plate,  Fig.  66,  which  can  be  placed  at  c  of 


THE  MICRO-SPECTROSCOPE.  149 

Fig.  57  ;  so  that  light  from  the  mirror  F  will  pass  through  the 
combination  before  entering  c  and  will  be  reflected  upward  by 
the  comparison  prism  D  to  the  eye  at  K.  The  half  of  the  field 
of  view  which  pertains  to  the  comparison  prism  will  be  occu- 
pied therefore  by  an  image  of  the  standard  scale,  with  its  twelve 
well  marked  divisions,  while  the  other  half  of  the  field  will 
show  in  direct  relation  to  this  the  spectrum 
bands  of  the  object  on  the  stage  of  the  micro- 
scope. The  standard  scale  H  shown  in  situ 
upon  the  binocular  spectroscope,  Figs.  61  and 
62,  is  a  very  compact  form  in  which  the  Nicol's 
prisms  are  replaced  with  thin  plates  of  iodide 
of  disulphate  of  quinine  known  as  Herapathite.  Fig.  67  shows 
the  appearance  of  a  field  of  view  in  which  two  absorption  bands 
in  the  upper  half  of  the  field  are  being  located  by  means  of  the 
image  of  the  standard  scale  in  the  lower  half.  R.  H.  W.] 

F.  Using  the  Micro-spectroscope.  To  the  botanist  the  mi- 
cro-spectroscope is  an  apparatus  of  such  importance  that  it  indeed 
must  be  occasionally  used  by  every  one  who  shall  undertake 
the  thorough  microscopical  investigation 
of  plants.  A  few  remarks  therefore  con- 
cerning its  use  will  not  be  superfluous. 

The  objects  to  be  investigated  are  of 
two  sorts,  liquid  and  solid.  Coloring 
substances  as  chlorophyll,  phykophaein, 
etc.,  will  frequently  come  under  micro- 
spectroscopic  investigation  in  the  form  of 
a  solution.  In  general  we  need  scarcely 

say  anything  concerning  the  preparation  of  the  solution.  In 
reference  to  the  chlorophyll  of  the  phanerogams  especially,  the 
particular  part  of  the  plant  from  which  the  preparation  is  to  be 
made,  as  for  instance  the  foliage  leaves,  is  put  for  a  short  time 
in  boiling  water,  then  quickly  dried  by  means  of  bibulous  paper 
and  then  immersed  for  a  longer  time  in  absolute  alcohol,  ether 
or  benzole,  in  a  dark  place,  for  the  purpose  of  extracting  the 
chlorophyll  coloring  matter.  The  concentration  of  the  solution 
thus  produced,  which  influences  the  intensity  of  the  absorp- 
tion spectrum  and  the  number  and  length  of  the  absorption 


150 


THE  MICROSCOPE  IN  BOTANY. 


bands,  depends  naturally  upon  the  time  during  which  the  material 
is  in  the  extracting  medium,  as  well  as  on  the  quantity  of  the 
material.  Commonly  also  a  solution  of  less 
concentration  will  give  the  same  intensity  of 
spectrum  if  a  sufficiently  thick  layer  of  it 
be  used.  The  solution  can  commonly  be  ex- 
amined in  an  ordinary  test  tube.  The  test 
tube  is  filled  and  carefully  corked  and  then 
laid  on  the  stage  of  the  microscope,  or  held 
before  the  opening  of  the  comparison  prism 
as  the  case  may  be.  For  the  latter  purpose 
(bringing  liquids  before  the  opening  of  the 
comparison  prism),  a  small  open  trough,  made  of  zinc,  to  which 
are  cemented  two  parallel  glass  plates,  as  shown  in  Fig.  68,  is 
very  useful.  For  exact  investigations,  however,  the  trough 
flask  illustrated  in  Fig.  69  is  preferable.  It  is  a  flask,  whose 
two  sides,  back  and  front,  are  parallel,  furnished  with  a  careful- 
ly fitted  ground-glass  .stopper.  It  should  be  filled  quite  full 
of  the  solution  and  then  laid  with 
its  broad  side  on  the  stage.  It 
is  especially  indispensable  when 
we  wish  to  study  the  combination 
spectrum  of  two  solutions.  In 
that  case  two  flasks  are  filled 
each  with  a  different  solution 
and  both  laid  upon  the  stage  one 
upon  the  other. 

[For  the  purpose  of  examin- 
ing small  quantities  of  any 
liquid,  a  sufficient  depth  being 
obtained  with  very  little  ma- 
terial, vertical  glass  tubes  at- 
tached to  horizontal  plates  are 
used,  as  proposed  by  Mr.  Sorby 
and  shown  in  Fig.  70.  The  narrow  tubes  are  made  of  various 
lengths  in  order  to  present  different  thicknesses  of  the  con- 
tained fluid ;  the  broad  tube  being  higher  on  one  side  than 
the  other  and  thus  constituting  a  wedge-shaped  cell,  which 


FIG.  G9. 


INFLUENCE  OF  THE  SLIT  ON  THE  SPECTRUM.  151 

when  filled  and  closed  by  a  thin  cover-glass  will  present  a  vary- 
ing thickness  of  fluid  for  study  and  comparison.     R.  H.  W.] 

If  the  object  to  be  investigated  is  not  a  solution  but  a  prep- 
aration of  the  kind  which  we  commonly  employ  in  microscop- 
ical inquiries,  we  must  first  of  all  bring  it  into  the  focus  of  the 
objective-system.  To  do  this  we  must  first  remove  the  tube 
bearing  the  prisms,  open  the  slit  somewhat  and  use  the  appara- 
tus as  a  simple  ocular.19  If  one  has  to  deal  with  a  small  object 
which  would  not  entirely  fill  the  slit,  but  so  that  rays  of  light 
might  come  in  by  it  and  disturb  the  spectrum,  he  should  turn 
the  comparison  prism  so  as  to  shut  up  some  of  the  slit  with- 
out, however,  letting  in  the  light  upon  it,  and  then  bring  the 
object  up  near  to  it  and  from  the  other  side  shove  up  the  short- 
ening apparatus  as  close  as  is  necessary.  On  the  other  hand, 


FIG.  70. 

should  the  object  consist  of  a  number  of  single  minute  grains, 
which  would  cause  to  be  drawn  across  the  spectrum,  in  the  direc- 
tion of  its  length,  perpendicular  to  the  Fraunhofer  lines,  a  like 
number  of  dark  lines,  one  must  adjust  the  microscope  so  that 
the  object  will  be  a  little  out  of  focus,  somewhat  above  or  below 
the  true  focus.  In  this  way  we  shall  get  a  uniform  spectrum.20 
[The  spectrum  can  also  be  improved  in  some  other  cases  by 
likewise  throwing  the  object  somewhat  out  of  focus.  R.H.  W.] 
T/te  Influence  of  the  Slit  on  the  Spectrum.  In  the  investi- 
gation of  homogeneous  solutions  it  is  by  no  means  a  matter  of 
indifference  what  width  we  give  to  the  slit,  as  will  be  clear  from 
the  following  considerations.  S  8,  Fig.  71,  is  the  slit  of  a  spec- 
tro-microscope,  P  the  refracting  prism,  and  W  a  screen  which 
will  intercept  the  spectrum  produced  by  P.  The  slit  S  S  shall 
be  wide  enough  to  afford  entrance  to  the  three  rays  of  white 

19  Be  careful  not  to  open  it  too  wide,;else  in  closing  it  again  one  might  easily  get  dirt  on 
its  edges. 

20  See  G.  Kraus,  Zur  Kenntniss  der  Chlorophyll  farbstoffe,  Stuttgart,  1872,  p.  12,  /. 


152 


THE   MICROSCOPE   IN  BOTANY. 


light  abc.  The  ray  a  will  be  separated  into  its  elements  by 
P  and  produce  the  spectrum  a'  at  W.  Likewise  b  produces  bf 
and  c  the  spectrum  c'.  The  three  spectra  in  their  middle  parts, 
green  and  yellow,  overlap  each  other  and  produce  here  a  great 
number  of  mixed  colors.  But  had  the  slit  been  narrowed  by 
the  distance  cb  then  c  would  not  have  entered  and  the  spectrum 
c'  would  not  have  been  produced,  and  the  number  of  mixed 
color  in  the  middle  would  have  been  also  less.  Therefore  the 

narrower  the  slit,  the  purer 
and  richer  will  be  the  colors 
of  the  spectrum.  By  opening 
the  slit  wider  the  spectrum 
will  indeed  be  brighter,  but 
it  will  be  pure  only  at  the 
red  and  blue  ends  while  in  the 
middle  there  will  be  a  preva- 
lence of  compound  mixed 
light  from  all  the  possible 
beams,  and  here  the  Fraun- 
hofer  lines  cannot  be  seen. 
It  is  of  the  first  importance  not  only  to  devote  attention  to 
the  width  of  the  slit,  but  also  to  keep  the  edges  of  it  absolutely 
clean.  For  if  the  smallest  particles  of  dust  get  upon  them  they 
will  show  themselves  in  the  spectrum  by  a  like  number  of  black 
lines  drawn  over  its  whole  length  which  very  greatly  disturb 
the  observation.  As  we  have  explained,  a  pinion  with  a  milled 
head  serves  to  adjust  the  ocular  lens  to  the  slit  and  the  Fraun- 
hofer  lines.  The  adjustment  differs  with  different  eyes.  By 
turning  the  milled  head  back  and  forth,  the  ocular  is  so  placed 
that  the  slit  will  appear  sharp,  and  when  daylight  is  used,  the 
Fraunhofer  lines  will  appear  with  the  greatest  possible  sharp- 
ness and  distinctness. 

If  the  apparatus  is  a  good  one  a  great  number  of  these  lines 
may  be  seen.  They  furnish  the  best  test  of  the  performing 
qualities  of  the  apparatus.  We  give,  in  Fig.  72,  an  illustration 
of  a  spectrum  made  from  common  daylight  by  means  of  Sei- 
bert's  spectroscope  into  which  the  visible  Fraunhofer  lines 
between  D  and  F  are  brought.  Their  distances  apart  have  been 


FIG.  71. 


INFLUENCE  OF  THE  SLIT  OX  THE  SPECTRUM. 


153 


determined  by  the  measuring  apparatus  constructed  by  the  same 
firm. 

Suppose  now  we  had  for  examination  the  spectrum  of  some 
matter,  for  example  of  a  kind  of  chlorophyll  from  the  leaves  of 
Primula  (after  Kraus).  We  should  observe  in  this  spectrum  : 

(1)  The  number  of  the  absorption  bands. 

(2)  Their  position  and  breadth. 

(3)  Their  relative  brightness. 


1 


I 


1L 


FIG.  7-2. 

The  number  of  the  absorption  bands  can  evidently  be  found 
by  counting.  Their  position  can  be  determined  in  two  ways. 
First,  one  can  determine  this  approximately  by  fixing  in  the  eye 
the  Fraunhofer  lines  and  estimating  the  position  of  the  bands 
in  relation  to  them.  The  breadth  may  be  obtained  in  the  same 
way  by  estimating  their  distance  from  two  lines  in  the  neigh- 
borhood. But  this  method  will  yield  only  approximate  results. 


Yl 


Ytt 


FIG.  73. 


If  one  would  be  exact  he  must  have  recourse  to  the  measuring 
apparatus.  The  measuring  is  done  by  moving  the  triangle  over 
the  whole  length  of  the  spectrum  and  noting  at  the  beginning 
and  end  of  each  band  the  position  of  the  index.  It  is  much 
more  difficult  to  estimate  the  relative  brightness  of  the  absorp- 
tion bands  since  here  we  bring  to  bear  a  subjective  judgment 
only.  In  the  case  before  us  we  come  to  the  conclusion  that  the 
absorption  bands,  B  C  (Band  I,  Fig.  73)  and  F  (Band  Y)  are 


154 


THE  MICROSCOPE  IN  BOTANT. 


the  darkest,  the  spectrum  colors  are  at  these  points  most  per- 
fectly absorbed.  The  bands  II,  VI  and  VII  are  of  medium 
brightness,  while  the  two  bands  III  and  IV  are  the  brightest  of 
all.  There  are  no  other  mechanical  aids  for  these  determi- 
nations at  the  present  time.  A  micro-spectre  photometer  is 
not  known  to  us. 

Since  measuring  the  breadth  and  position  of  the  absorption 
bands  often  consumes  much  time,  the  comparison  of  spectra 
may  be  employed  with  advantage,  when  the  spectrum  of  the 
object  being  investigated  seems  to  be  like  that  of  one  already 
known  to  us.  We  put  the  material  under  investigation  (we 
will  suppose  a  solution)  in  a  trough  on  the  stage,  and  the 
known  solution  before  the  opening  of  the  comparison  prism, 
and  shove  the  prism  into  place.  If  then  the  position  and  breadth 


FIG.  74. 


of  the  bands  are  alike  it  is  sufficient  grounds  for  assuming  the 
solutions  to  be  identical.  If  the  material  is  not  identical  but 
related,  then  the  differences  in  the  spectra  will  be  small,  and  can 
be  easily  indicated  on  paper  by  means  of  the  measuring  apparatus. 
We  see  illustrated,  for  example,  in  Fig.  74,  I,  the  well  known 
spectrum  of  oxyhamoglobin.  With  this  is  the  spectrum  II,  of 
a  substance  (hamoglobin,  reduced  hamoglobin)  for  compari- 
son, which  is  produced  by  giving  to  the  former  solution  some 
drops  of  the  solution  of  ammoniacal  iron  tartrate. 

We  will  consider  here  only  the  absorption  bands  between  D 
and  E.  We  need  not  measure  the  darkest  part  of  the  absorp- 
tion band  of  II  since  it  lies  exactly  between  the  two  dark  bands 
of  the  comparison  spectrum.  This  is  necessary  only  for  the 
outer  borders  of  the  brighter  absorption  stripes.  For  this  pur- 
pose the  measuring  triangle  must  be  focussed  on  the  dark  line 


INFLUENCE  OF  THE  SLIT  ON  THE  SPECTRUM.      155 

which  separates  the  spectra  and  then  determine  the  distance 
from  the  edges  of  the  absorption  stripes  in  II  to  the  nearest 
lying  edges  of  those  in  I,  Fig.  74. 

Should  we  wish  to  examine  the  spectra  of  the  same  fluid  in 
layers  of  different  thicknesses,  we  might  put  it  into  long  glass 
tubes  which  have  been  melted  off  even  at  the  bottom,  and  which 
may  be  shoved  into  the  tube  of  the  microscope  when  the  objec- 
tive has  been  removed,  according  to  Pringsheim's  method. 
Then  by  gradually  filling  the  tube  with  the  solution  one  can 
easily  observe  the  changes  which  take  place  in  the  spectrum.21 

21  For  the  technique  of  micro-spectroscopical  investigations  one  msy  consult:  G.  Kraus, 
Znr  Kenntniss  der  Chlorophyllfarbstoffe,  Stuttgart,  1872.  Pringsheim.  Ueber  die  Absorp. 
tionspectra  der  Chlorophylllarbstoffe  (Monatsber.  der  K.  Academic  der  Wissenschaften, 
zu  Berlin,  Oct.,  1874,  pp.  028-059,  nebst  1  Tfl.).— Xageliund  Schwendener,  Das  Mikroskop, 
pp.  436-440. 


CHAPTER  III. 
THE  PREPARATION  OF  MICROSCOPICAL  OBJECTS. 

I.     INTRODUCTION. 

ALTHOUGH  there  are  many  opportunities  to  buy  ready  made 
microscopical  preparations,  still  every  one  who  uses  the  mi- 
croscope for  something  more  than  amusement  is  compelled  so 
to  prepare  those  parts  of  plants  which  he  is  to  examine  that 
they  will  be  suitable  for  microscopical  research.  And  it  will 
often  be  desirable  to  prepare  the  object  to  be  investigated  so 
that  it  may  be  preserved  for  any  length  of  time,  and  placed 
at  any  moment  under  the  microscope.  There  has  therefore 
sprung  up,  chiefly  in  recent  times,  a  microscopical  technique, 
to  learn  which  indeed  requires  a  pretty  long  schooling,  but 
which  may.be  learned  by  anyone  if  he  does  not  lack  the  neces- 
essary  time  and  perseverance.  For  those  especially  who  have 
no  patience,  the  preparation  of  microscopical  objects  is  the 
best  means  of  attaining  it.  But  those  who  have  patience  will 
often  enough  lose  it  in  this  employment.  One  must  not  be 
afraid  of  making  failures  at  first,  even  though  they  should  be 
repeated  many  times,  but  remember  that  perseverance  always 
leads  on  to  success. 

We  remarked  that  microscopical  technique  had  been  built  up 
in  recent  times.  Formerly  it  was  thought  that  if  a  part  of  a 
plant  needed  anything  to  be  done  to  it  before  putting  it  under 
the  microscope,  it  was  sufficient  simply  to  crush  it.  Sachs  has 
mentioned  in  his  "History  of  Botany"1  the  sad  influence, 
upon  the  development  of  vegetable  anatomy,  of  this  rough 
method  of  preparation  which  prevailed  during  the  whole  of  the 
last  century.  It  was  in  the  first  third  of  our  century  that  Hugo 
v.  Mohl  undertook  to  bring  the  preparation  of  microscopic 
objects  to  the  excellence  which  they  have  reached  in  our  time. 


(156) 


INTRODUCTION.  157 

He  first  emphasized  the  advantage  of  immersing  every  object 
which  was  to  be  submitted  to  microscopical  investigation,  as 
far  as  possible,  in  some  fluid.  He  first  introduced  the  cover- 
glass  into  general  use. 

Of  almost  still  more  recent  date  is  the  preparation  of  those 
objects  which  are  to  be  preserved  for  a  long  time  and  are  called 
permanent  preparations.  Who  has  not  seen  lying  by  old  mi- 
croscopes wooden  strips  with  a  series  of  holes  in  them,  in  which 
two  round  glasses  were  clamped  by  means  of  an  elastic  metal 
ring,  bearing  between  them  the  preparations  (1)  a  fly's  wing, 
(2)  a  leaf  of  moss,  (3)  a  human  hair,  (4)  a  spider's  foot,  (5)  a 
linen  thread,  (6)  the  best  of  all,  a  flea?2  How  lamentable  was 
the  case  in  respect  to  the  preparation  of  permanent  mounts 
even  down  to  Hugo  v.  Mohl's  time,  one  may  best  get  an  idea 
by  reading  through  the  conclusion  of  his  "Mikrographie"  of 
1846. 

He  proposed  that  the  object,  in  case  of  a  dry  preparation, 
should  be  placed  between  two  slips  of  mirror  glass  (of  the  size 
of  our  slides)  and  in  order  to  keep  the  dust  out  to  paste  the 
edges  about  with  strips  of  paper.  If  the  object  were  to  be 
preserved  in  a  moist  condition,  a  drop  of  the  solution  of  chlor- 
ate of  lime  was  placed  between  the  glasses,  the  object  immersed 
in  it,  and  inclosed  by  two  paper  slips  smeared  with  a  thick  rub- 
ber gum  solution  and  put  across  between  the  glasses.  No  one 
nowadays  thinks  of  preserving  objects  in  this  way.  We  shall 
see  presently  that  we  have  now  other  and  better  methods  of 
preparation. 


It  is  necessary,  in  the  first  place,  to  know  exactly  how  a  mi- 
croscopical preparation  should  be  made  so  as  to  satisfy  all 
requirements.  , 

The  first,  and  in  all  cases  the  same  requirement,  is  that  the 
part  of  the  plant  under  consideration  shall  be  by  nature,  or  shall 
be  made,  so  thin  that  light  may  go  unhindered  through  it,  so 

2  In  the  catalogue  of  Leeuwenhoek's  collection  of  microscopical  preparations,  which 
has  been  preserved,  occur,  "a  hair  from  the  noee,"  and  ''oysters  not  yet  hatched  out  in 
a  small  tube." 

'P.328,/. 


158  THE  MICROSCOPE  IN  BOTANY. 

that  it  shall  be  perfectly  transparent.  The  examination  of  opaque 
objects  by  reflected  light  does  not  occur  so  far  as  I  know  in 
vegetable  anatomy.  The  preparation  should  also  exhibit 
the  parts  investigated  in  their  natural  and  undisturbed  con- 
dition. Objects  which  indeed  are  thin  enough,  but  whose 
individual  parts  are  separated  from  each  other  and  torn,  are 
commonly  not  in  a  condition,  for  satisfactory  examination. 
Finally,  objects  must  be  examined  in  a  medium  (fluid),  which 
will  allow  the  structure  to  be  seen  in  the  most  natural  possible 
condition.  I  say  in  the  most  natural  possible  condition  because 
there  is  no  fluid  known  perhaps  which  will  show  the  microscopic 
object  altogether  naturally. 

The  fluid  applied  to  the  object  may  impart  fluid  to  it,  or 
withdraw  fluid  from  it.  In  the  first  case  the  organ  will  swell 
and  in  the  second  it  will  shrink.  If,  for  example,  one  puts 
chlorophyll  or  starch  grains  into  a  solution  of  calcium  chlorate, 
they  will  immediately  swell,  while  cell  walls  remain  quite  un- 
changed in  it,  but  the  latter  immediately  swell  if  they  be  put 
into  a  concentrated  solution  of  chlor-iodide  of  zinc.  Potassium 
bichromate  and  chromic  acid,  also  alcohol  and  glycerine, 
often  produce  a  shrinking,  and  the  latter  always  contracts  the 
primordial  article.  One  might  perhaps  think  that  water  would 
act  indifferently  upon  the  preparation,  but  this  is  by  no  means 
the  case.  Gum  arabic  immediately  swells  up  in  water  and 
becomes  a  mucilage,  likewise  all  muciparous  cell  walls  are  thus 
immediately  changed  in  this  medium,  by  considerable  swelling. 
One  must  in  each  case  choose  the  right  medium  for  the  investi- 
gation and  will  get,  for  example,  a  much  more  natural  image  of 
the  muciparous  celUwalls  in  absolute  alcohol  than  in  water. 

It  is  well  known  that  fluids  possess  a  much  greater  refractive 
power  than  the  air.  We  express  the  magnitude  of  this  by  means 
of  the  refractive  index  or  the  exponent  of  refraction,  that  is  to  say, 
in  the  passage  of  light  from  the  air  into  a  given  fluid,  there  is 
a  constant  relation  between  the  sine  of  the  angle  of  incidence 
and  the  sine  of  the  angle  of  refraction.  The  numerical  expres- 
sion of  this  relation  is  the  refractive  index  or  exponent.  Thus 
the  exponent  of  refractio.i  of  some  of  the  more  important  fluids, 
suitable  for  microscopical  use,  is  as  follows  : 


REFRACTIVE  POWER  OF  FLUIDS.  159 


Anise  oil, 
Tolu-balsam, 
Cassia  oil, 
Canada  balsam, 
Citron  oil, 
Turpentine  oil, 
Pure  glycerine, 
Olive  oil, 


.811  Sulphuric  acid,  1.430 

.628-  Glycerine  and  water 

.610  (equal  parts),  1.400 

.530  Glacial  acetic  acid,  1.380 

.527  Alcohol,  1.370 

476  Ether,  1.360 

475  Albumen,  1.350 

470  Water,  1.336 


It  is  now  a  well-known  fact  that  an  object  becomes  more 
distinctly  visible,  the  more  its  refractive  power  differs  from  that 
of  the  medium  in  which  it  is  mounted.  We  have  already  seen 
that  it  is  not  a  matter  of  indifference  how  a  specimen  is  mounted 
which  is  to  be  brought  under  observation,  as  in  the  case 
of  the  test  objects  (butterflies'-scales,  diatoms,  etc.),  whether 
mounted  dry  and  so  surrounded  by  air  only,  or  lying  in  Canada 
balsam.  In  the  former  case  their  delicate,  interesting  markings 
are  much  more  easily  seen  than  in  the  latter.  Very  delicate 
plant  structure  is  much  more  difficult  to  recognize  in  glycerine 
than  in  water.  It  is  therefore  recommended  that  the  fluids  used 
in  preparing  objects  for  observation  be  such  as  have  the  least 
possible  refractive  power. 

But,  if  for  any  reason,  the  object  be  too  opaque  for  micro- 
scopical observation,  then  the  greater  refractive  power  of  the 
fluid  serves  as  an  important  means  for  rendering  it  trans- 
parent, and  brings  to  view  details  which  in  water  or  fluids  of 
like  refractive  exponent  would  not  be  seen.  The  beginner  will 
most  easily  see  the  clarifying  power  of  fluids  of  different  re- 
fractive index  if  he  will  examine  the  large  pollen  grains  (of 
Epilobium,  Cucurbeta,  Malva,  etc.),  first  in  alcohol,  then  in 
Canada  balsam  and  in  anise,  or  clove  oil.  If  in  practice  one 
wishes  to  find  a  fluid  of  most  suitable  clarifying  powers  for  a 
preparation,  it  may  be  found  most  conveniently  in  the  table 
just  now  given. 

How  very  much  the  refractive  power  of  the  mounting  fluid 
determines  the  visibility  of  a  microscopic  object  is  illustrated 
by  this  experiment.  A  fine  glass  rod  lying  in  water  is  very 
easily  recognized  by  reason  of  the  difference  of  their  exponents 
of  refraction.  But  if  we  lay  it  in  Canada  balsam,  the  exponent 
of  which  is  very  nearly  the  same  as  that  of  the  rod,  the  rod  will 


160  THE  MICROSCOPE  IN  BOTANY*. 

cease  to  glitter  mid  it  will  be  only  by  giving  the  closest  atten- 
tion that  we  can  distinguish  it,  and  then  as  a  flat  band.  But 
if  it  be  put  in  anise  oil  we  get  an  image  of  it  as  if  it  were  a 
a  hollow  space  run  through  the  oil.  (Welcker.)4 


II.     THE  PREPARATION  OF  OBJECTS  WITHOUT 
CUTTING  INSTRUMENTS. 

Except  in  rare  cases  microscopic  objects  will  be  obtained 
by  dissecting  out  the  parts  of  the  plant  with  a  knife.  To 
these  exceptional  cases,  however,  belong  all  such  plants  and 
plant  organs  as  are  so  thin  that  they  may  be  laid  tinder  the  micro- 
scope without  further  manipulation,  and  also  those  fragments  of 
thicker  parts  which  one  may  obtain  by  maceration  or  incinera- 
tion. 

A.    OBJECTS  FOR  IMMEDIATE  OBSERVATION. 

As  such,  we  name  first  the  delicate  hairs  of  the  higher  plants, 
next  those  parts  of  the  cuticle  which  are  easily  torn  away,  then 
those  leaves  of  mosses  and  liverworts  which  consist  of  a  single 

O 

layer  of  cells,  and  finally  a  great  number  of  algre  and  fungi. 

Delicate  hairs  only  need  to  be  separated  from  the  plant  by 
means  of  a  sharp  knife,  or  torn  off  with  sharp  forceps  and  put 
in  water  or  glycerine  to  be  immediately  subjected  to  investiga- 
tion. In  this  way,  for  instance,  we  obtain  the  beautiful  prepar- 
ation which  shows  the  circulation  of  the  protoplasm  in  the 
staminate  hairs  of  Tradescantia,  or  in  the  root  hairs  of  Hydroch- 
aris  Morsus  raitce.  Likewise  in  this  way,  a  piece  of  the  epi- 
dermis of  higher  plants  may  be  immediately  examined  which 
one  may  tear  away  from  the  leaves  with  the  forceps.  The 
leaves  of  many  monocotyledons  (Lucojum,  Galantlnis,  Hya- 
cinthus,  Orchis),  lend  themselves  to  this  procedure  most  readily. 
The  leaves  of  mosses  and  liverworts  may  be  broken  from  the 
mother  plant  with  the  forceps  and  immediately  immersed  in  a 
drop  of  water  and  laid  under  the  microscope. 

4  Frey,  Das  Mikroskop,  Leipzig,  1877,  p.  73. 


OBJECTS  FOR  IMMEDIATE  OBSERVATION.  161 

A  great  number  of  the  lower  thallophytes  may  be  subjected 
to  examination  without  previous  preparation.  Of  the  mauy- 
celled  plants  belonging  to  this  group,  are  the  Hydrodictyeae, 
Ulotrichaeeae,  Zygmaceae,  Mucorineae,  Piptocephalideae,  Sphae- 
ropleae,  Oedogoniaceae,  Confervaceae,  many  Ulvaceae,  Coleochae- 
teae,  and  many  others,  and  the  great  number  of  the  single-celled 
thallophytes  including  all  the  swarm  spores. 

The  single-celled  organisms,  on  account  of  their  almost  or 
quite  microscopical  minuteness,  are  very  difficult  to  bring  into 
the  examining  drop  under  the  microscope ;  in  many  cases  it  is 
accomplished  only  by  a  fortunate  accident.  The  larger  of  them 
however  may  be  best  found  by  the  following  simple  means  first 
suggested  by  Ehrenberg.  Fill  a  wide  watch-glass  with  a  por- 
tion of  the  water  which  we  suppose  contains  the  larger  forms 
ot  the  single-celled  algae,  and  place  it  on  a  piece  of  white  paper 
which  has  had  one-half  blackened  with  India  ink,  so  that  the 
background  of  one-half  of  the  glass  is  black  and  the  other  half 
white.  On  the  dark  background  the  light  colored  and  on  the 
white  the  dark  colored  plants  become  conspicuous.  Examine 
the  glass  now  with  a  not  too  weak  magnifier  and  we  shall 
easily  distinguish  the  principal  forms.  In  this  manner  I  have 
always  easily  recognized  the  species  of  Pediastrum,  Closterium, 
Pandorina  morum  and  many  others ;  indeed,  of  the  first  genus, 
approximately  to  guess  the  species. 

We  often  discover  a  desired  species  in  a  test  tube  of  water 
containing  algae,  and  wish  to  subject  it  to  observation.  AVe 
may  best  do  this  by  taking  a  glass  tube,  evenly  cut  off  at  both 
ends,  of  about  2-3  mm.  interior  diameter  and  closing  one 
end  of  it  with  the  index  finger  of  the  right  hand,  put  the 
other  end  slowly  down  into  the  water.  The  inclosed  air  keeps 
the  water  out.  Xow  bring  the  lower  open  end  of  the  tube 
directly  over  the  floating  alga,  and  suddenly  remove  the  finger 
from  the  upper  end.  The  water  rushes  into  the  tube  with  great 
force  —  all  the  more  forcibly  the  farther  beneath  the  surface  of 
the  water  the  lower  end  of  the  tube  is  —  and  carries  with  it  all 
the  objects  swimming  in  the  neighborhood  and  our  alga  among 
them.  The  tube  should  then  be  again  quickly  closed  at  top 
and  withdrawn  from  the  water.  Its  contents  should  be  poured 
11 


162  THE  MICROSCOPE  IN  BOTANY. 

into  a  watch-glass,  and  here,  in  this  limited  space,  the  hunt  for 
the  alga  may  be  made.  Or,  if  one  can  see  the  alga  in  the  tube, 
by  partly  opening  the  upper  end,  the  water  may  be  suffered  to 
run  out  slowly,  drop  by  drop,  till  the  alga  has  come  clown  into 
the  underhanging  drop,  when  it  may  be  quickly  captured  and 
removed  with' a  slide.  Still  better,  by  means  of  an  apparatus, 
illustrated  in  Fig.  75,  the  drop  with  the  alga  in  it  may  be 
brought  directly  upon  the  slide.  This  contrivance  consists  of 


FIG.  75. 

a  glass  tube  narrowed  at  the  lower  end  and  widened  into  a 
hemispherical  form  at  the  top,  covered  with  a  small  piece  of 
rubber.  Its  use  is  understood  without  further  explanation.5 

B.    MACERATING  OB  SOFTENING. 

This  method  of  preparation  depends  on  the  fact  that  plant 
tissues,  which  have  lain  in  a  certain  fluid  for  a  long  time, 
undergo  in  part  a  disintegration,  while  other  parts  resist  the 
destructive  influences  and  so  are  parted  from  each  other,  and 
thus  separated  are  fit  for  examination. 

B  In  along  note  the  author  gives  particular  directions  for  "blowing"  these  pipettes,  for 
one's  self,  out  of  glass  tubing.  But  it  seems  scarcely  necessary  to  do  that  when  one  can 
buy  them  very  cheaply  of  the  dealers,  or  may  substitute  "dropping  tubes"  which  may  be 
had  at  any  apothecary  shop  for  a  few  cents.  A.  B.  H. 


MACERATING  OR  SOFTENING.  163 

Very  delicate  plant  tissue  may  be  macerated  by  lying  for  a 
considerable  time  in  distilled  water.  But  the  process  may  be 
greatly  hastened  by  warming  the  water.  In  this  way  one  may 
obtain  the  large  continuous  epidermis  of  many  foliage  leaves, 
particularly  of  the  above  named  monocotyledons.  Finally,  other 
soft  parts  of  plants  may  be  macerated  by  continual  boiling  in 
water.  Hartig6  recommends  a  well  tinned  vessel  into  whose 
closely  fitting  cover  is  soldered  a  glass  tube,  a  meter  long,  much 
inclined  from  the  perpendicular.  The  steam  condenses  in  the 
glass  tube  and  runs  back  into  the  vessel  so  the  boiling  may  go 
on  as  long  as  it  is  necessary  without  particular  care. 

A  process  for  macerating  the  woody  organs  of  plants  has 
been  given  by  M.  Schultze,  and  consists  of  treating  the  tissue 
to  be  macerated  with  potassium  chlorate  and  nitric  acid.  The 
tissue  is  boiled  some  seconds  in  a  flask  with  nitric  acid  to  which 
is  added  a  little  potassium  chlorate.  Then  pour  the  whole  out 
into  a  considerable  quantity  of  pure  water  and  fish  out  the  plant- 
tissue  with  a  little  glass  slip,  and  it  can  then  easily  be  dissected 
with  a  couple  of  needles  (Hartig).7  Hartig8  puts  small  pieces 
of  the  cellular  tissue  to  be  treated  in  a  test  tube  with  a  like 
volume  of  potassium  chlorate,  then  pours  over  it  concentrated 
nitric  acid,  and  warms  over  the  spirit  flame  to  boiling,  till  the 
cells  separate  and  then  washes  out  with  water.  It  is  scarcely 
necessary  to  mention  that  by  this  process  all  cell  substances 
with  the  exception  of  some  cell  walls  are  destroyed.9 

According  to  Hartig10,  artificial  freezing  mixtures  furnish  a 
good  means  of  separating  into  their  elements  the  cellular  tis- 
sue of  ripening  or  germinating  seeds.  A  good  freezing  mix- 
ture is  produced  by  pulverized  glauber  salts  and  hydrochloric 
acid  by  which  a  refrigeration  of — 12°  without  special  pains  and 
in  high  summer  temperature  is  easily  obtained.  The  object 
should  be  put  in  a  thin-walled  test  tube  and  left  in  the  cold  mix- 
ture as  long  as  the  temperature  is  below  zero. 

6  Hartig,  Entwicklnngsgeschichte  des  Pflanzenkeimes,  Leipzig,  1858,  p.  153. 

7  Havtig,  Das  Mikroskop,  p.  394. 

8  Hartig,  1.  c.,  p.  153. 

«  Kabsch  in  Pringsheim's  Jahrb.,  Bd.  Ill,  p.  357. /. 
10  Havtig,  1.  c.,  p.  153. 


164  THE  MICROSCOPE  IN  BOTANY. 


C.    INCINERATING-  AND  CALCINING. 

It  is  well  known  that  in  the  superficial  layer  of  many  plants 
there  is  intercalated  a  silicate,  by  which  a  considerable  rigidity 
is  imparted  to  it.  Since  the  silicate  is  not  destroyed  by  burning 
or  by  mineral  acids  it  may  be  easily  obtained  in  the  form  of  a 
siliceous  skeleton,  after  the  more  delicate  parts  of  the  tissue 
have  been  destroyed.  For  this  purpose  treat  the  given  tissue 
(for  example,  a  piece  of  the  epidermis  of  .Equisetum  hiemale), 
first,  with  Schultze's  macerating  mixture  till  it  is  colorless, 
wash  it  well  and  then  burn  out  the  residuum  on  platinum  foil.11 
The  siliceous  skeletons  of  the  Diatomaceae  may  be  prepared  in  a 
pure  state  by  this  process.  Sachs 12  employs  the  following 
method  for  incineration.  "In  order  to  obtain  beautiful  skele- 
tons it  is  necessary  to  soak  the  detached  epidermis,  or  thin 
section  beforehand,  in  nitric  or  muriatic  acid,  and  then  burn  it 
on  the  platinum  foil.  I  have  found  another  method  still,  much 
more  convenient.  I  lay  larger  pieces  of  the  tissue  (for  ex- 
ample, grass  blades,  equisetum  stems,  etc.)  on  the  platinum 
foil  in  a  large  drop  of  concentrated  sulphuric  acid  and  heat  it 
over  the  flame.  The  mass  at  once  turns  black  and  a  powerful 
development  of  gas  takes  place.  The  heating  should  continue 
till  a  pure  white  ash  alone  remains.  This  very  soon  happens, 
while  the  common  method  of  incineration  almost  always  takes 
a  good  deal  of  time  and  does  not  always  produce  a  perfectly 
colorless  skeleton." 

Many  cells  contain  calcareous  matter  inlaid  or  onlaid,  making 
them  opaque  and  unfit  for  microscopical  investigation.  In  many 
cases  the  mineral  matter  (if  it  be,  as  is  common,  calcium  car- 
bonate) may  be  removed  by  treatment  with  dilute  muriatic  acid 
(species  of  Cham,  Corallines,  etc.).  For  this  purpose  put  the 
section  or  the  whole  of  the  plant,  as  the  case  may  be,  for  a 
longer  or  shorter  time  in  cold  acid  which  dissolves  the  incrust- 
ing  calcium  with  a  development  of  carbonic  acid. 

«  Hugo  v.  Mohl  in  Botan.  Zeitung,  1861,  p.  208. 
«  Sachs'  Lehrbuck  der  Botanik,  III,  Auflage,  p.  38. 


INSTRUMENTS  FOR  MICROSCOPIC  THIN  SECTIONS.         165 


III.     INSTRUMENTS  FOR  THE  PREPARATION  OF 
MICROSCOPIC  THIN  SECTIONS. 

With  the  exception  of  the  infrequent  cases  already  mentioned 
the  microscopist  will  be  constantly  confronted  with  the  task  of 
preparing  a  very  delicate,  perfectly  transparent  section  of  the 
organ  under  investigation.  For  this  purpose  different  instru- 
ments are  necessary,  and  the  success  of  the  preparation  mate- 
rially depends  upon  the  good  order  and  proper  management  of 
these  instruments. 

It  is,  therefore,  first  necessary  to  make  ourselves  acquainted 
with  the  tools  we  are  to  use  before  we  undertake  the  prepara- 
tion of  the  section  itself. 

It  was  formerly  the  opinion,  as  v.  Mohl  has  expressed  it  in. 
his  own  stiking  way,13  that  an  artificially  augmented  power  of 
vision  required  artificially  enhanced  capability  of  hands  —  that 
the  preparation  of  the  object  would  become  easy  by  help  of  a 
multitude  of  instruments  and  artificial  apparatus.  "  With  all 
these,  few  will  be  helped.  One  may  train  his  hand  to  surer  and 
steadier  motions  than  the  simplest  apparatus  can  accomplish. 
To  no  one  does  the  saying  of  Franklin  so  fittingly  apply  as  to 
the  microscopist.  'A  naturalist  must  be  able  to  saw  with  an 
auger,  and  bore  with  a  saw."3  We  must  apply  these  words  of 
the  great  anatomist  in  the  strictest  sense,  because  we  believe 
that  those  who  are  the  most  skilful  preparators,  and  who  attain 
the  best  results  are  those  who  understand  how  to  work  with  the 
fewest  and  simplest  instruments  possible.  Just  as  little  as  it  is 
possible  to  construct  a  machine  for  the  calculator — really  for 
many  very  desirable  —  which  shall  lead  him  mechanically,  with 
mathematical  certainty  to  right  conclusions,  even  so  little  is  it 
possible  to  invent  an  apparatus  which  in  the  hands  of  the  un- 
skilful and  inattentive  will  turn  out  objects  for  the  microscope 
suitable  for  investigation. 

The  instruments  commonly  employed  by  the  microscopical 

"  H.  v.  Mohl,  Mikvographie,  p.  255,  f. 


166  THE  MICROSCOPE  IN  BOTANY. 

preparator  are  the  razor,  scalpel,  lancet,  scissors,  needles,  and 
forceps.* 

A.  The  Razor.  Among  the  preparing  instruments  of  the 
microscopist  the  razor  occupies  the  most  prominent  place.  We 
employ  the  form  of  handle  prepared  for  the  barber's  uses,  with 
a  blade  which  is  movable  in  the  handle.14  The  requirements 
which  a  razor  should  satisfy  in  order  to  be  really  serviceable  are 
somewhat  the  following.  The  razor  should  be  strongly  made,  but 
at  the  same  time  relatively  as  light  as  possible.  A  knife  which 
has  a  heavy  handle  or  a  very  massive  blade  soon  tires  the  hand 
while  cutting.  Further,  the  heel  at  the  base  of  the  blade  should 
be  round  and  blunt  edged,  not  angular  and  sharp  cornered. 
This  —  though  it  is  not  commonly  thought  of — is  important, 
because,  in  cutting,  one  has  to  lay  the  end  of  his  thumb  on  the 
heel,  and  if  this  be  sharp  or  angular  it  will  after  a  while  scarcely 
fail  to  do  injury  to  the  skin  of  the  thumb.  Finally,  the 
blade  should  be  of  the  best  hardened  steel  and  as  broad  as 
possible. 

As  to  the  form  of  the  blade,  one  should  choose  two  kinds 
adapted  to  his  needs,  viz.,  first,  a  pretty  thick  blade  which  gives, 
in  section,  somewhat  the  form  shown  in  Fig.  76, 1.  It  is  ground 

but  slightly  hollowing  and  should  not 

?  spring  when  pressed    upon    the  finger 

W  nail.     For  the  second,  take  one  ground 

*  very  concave,  consequently  of  a  leaf- 
like  form  (section  seen  Fig.  76,  II), 
which  springs  when  one  presses  it  upon 
the  thumb  nail  and  rings  when  struck 
with  the  finger.  The  last  form  is  not 
commonly  furnished  by  the  instrument  makers,  but  can  be  ground 
out  from  the  first  named  form.  Finally,  there  is  a  form  recently 
introduced  in  which  the  blade  is  concave  on  the  upper  and 
plane  on  the  lower  side,  Fig.  76,  III.  It  is  capable  of  doing 

*  Also  some  other  tools  to  be  described  hereafter,  including  the  section  instrument  or 
microtome,  and  of  course  the  straight  edged  knife.  The  author  does  not  include  the  sec- 
tion cutter  but  it  will  be  found  extremely  useful  in  a  great  variety  of  cases,  and  in  many 
quite  indispensable.  The  instrument  and  its  use  will  be  found  described  farther  on.  A.B.H. 

14  Razors  are  furnished  by  some  microscopical  institutes  which  when  opened  the  blade 
may  be  made  fast  in  the  handle.  For  cutting  with  a  microtome  this  might  be  practicable,  but 
it  is  not  so  serviceable  in  conducting  free-hand  cuttings. 


INSTRUMENTS  FOR  MICROSCOPIC  THIN  SECTIONS.         167 

good  service  in  cutting  wood  sections,  but  has  the  fault  of  being 
very  difficult  to  sharpen. 

[The  J.  R.  Torrey  Razor  Co.,  of  Worcester,  Mass.,  regular- 
ly manufactures  razors  exactly  answering  to  the  description  of 
forms  I  and  II.  Their  No.  583  has  a  blade  of  the  form  indicated 
in  Fig.  76,  I,  and  their  No.  147  has  a  very  fine,  thin,  broad 
blade  with  a  sectional  view  exactly  corresponding  to  Fig.  76, 
II.  For  breadth,  finish  and  quality  of  blade  these  razors  leave 
nothing  to  be  desired,  and  probably  excel  those  of  foreign  make. 
This  firm  also  makes,  especially  for  naturalists'  use,  the  plano- 
concave razor  represented  in  Fig.  76,  III.  The  general  form 
of  this  razor  is  shown  in  Fig.  77.  The  side  not  shown  in  the 
illustration  is  the  one  that  has  been  flattened.  It  is  of  medium 
size,  considerably  smaller  than  the  others,  and  is  an  instrument 


FIG.  77. 

so  well  adapted  to  the  use  of  the  phytotomist  that  I  think  he 
will  employ  it  more  than  either  of  the  others,  not  only  for  mak- 
ing wood  sections  but  also  for  cutting  nearly  all  those  kinds  of 
soft  tissue  that  may  be  held  in  an  elder  pith,  and  for  this  reason. 
I  find  it  a  great  advantage  to  hold  the  elder  pith,  when  once  the 
section  material  is  rightly  embedded  in  it,  in  a  hand-vise.  Then 
by  so  holding  the  vise  in  the  left  hand  that  the  index  finger 
comes  to  a  position  partly  surrounding  the  pith  and  at  a  level 
with  the  top  of  it,  this  flat  side  of  the  razor  may  be  laid  on  the 
finger  in  such  a  way  as  to  make  of  it  a  very  excellent  rest  and 
guide  in  the  cutting,  giving  great  precision  to  the  movements 
of  the  knife  and  enabling  the  operator  to  cut  extremely  thin  and 
very  even  sections.  A.  B.  H.~] 


168  THE  MICROSCOPE  IN  BOTANY. 

The  razor  is  for  the  vegetable  anatomist  what  the  chisel  is  for 
the  sculptor  or  the  brush  for  the  painter.  He  needs  it  for  mak- 
ing almost  every  preparation,  and  he  must  therefore  devote  the 
greatest  attention  to  keeping  it  in  order. 

If  the  knife,  by  some  carelessness,  has  got  nicked,  the  sim- 
plest means  of  remedying  the  fault  is  to  take  it  to  the  instru- 
ment grinder.  But  the  right  kind  of  a  microscopist  will  not 
be  dependent  on  others  for  every  little  thing,  all  the  more  since 
by  a  little  pains  he  earn  remove  the  nick  himself.  For  this 
purpose  the  knife  should  be  applied  to  the  oil  stone  till  every 
part  of  the  fault  has  disappeared.  Five  or  ten  minutes  will 
commonly  be  required  for  this. 

The  oil  stone  is  a  whetstone  of  smoothly  polished  amorphous 
quartz,  the  best  and  hardest  being  brought  from  North  America.* 
A  drop  of  olive  oil  is  put  on  the  clean  stone,  the  razor  opened 
and  laid  flat  upon  the  stone,  back  and  edge  resting  upon  it, 
and  then  drawn  diagonally  over  the  stone  the  back  forwards.  On 
coming  to  the  upper  end  of  the  stone  turn  the  knife  over  on  its 
back,  and  draw  it,  back  forwards,  again  to  the  place  of  starting. 
This  should  be  repeated  as  long  as  it  is  necessary.  If  the  grind- 
ing is  being  rightly  accomplished  there  will  be  a  peculiar  sound  ; 
the  knife  must  be  "drawn"  over  the  stone  as  the  experts  say. 
When  by  continuous  work  on  the  oil  stone,  the  nicks  are  all 
removed,  the  oil  stone  is  to  be  replaced  by  a  softer  one  of  slate 
stone,  whereon  the  work  is  to  be  continued  in  the  same  manner 
only  with  the  use  of  water  instead  of  oil.15  We  now  have  an 
edge  without  a  nick,  but  not  one  quite  smooth.  One  may 
convince  himself  of  this  by  examining  it  with  a  magnifying 
glass,  when  he  will  see  that  all  along  the  edge  diagonally  to 
the  long  axis  of  the  blade  are  fine  furrows.  These  can  be  re- 
moved by  the  use  of  the  strop. 

Of  razor  strops  there  are,  as  is  well  known,  various  forms 
and  sizes.  There  are  also  very  bad  and  very  good  ones  ;  only  the 

*  The  Arkansas  oil  stone.    A.  B.  H. 

15  The  name  of  these  whetstones  is  not  known  to  me.  [They  are  sold  as  "  barbers' 
hones"  in  this  country.  A.  T5.  H.]  They  are  about  17.7  cm.  long  and  4  cm.  broad,  and 
consist  of  a  layer  of  oily-feeling  yellowish  stone  which  may  be  scratched  with  the 
knife  and  which  is  made  fast  to  a  piece  of  blue  slate  by  some  cementing  medium. 
They  are  to  be  had  in  the  larger  hardware  shops  for  about  1  Mark  apiece.  If  in  sharpening 
the  knives  the  stone  gets  scratched,  it  may  be  made  perfectly  smaoth  again  by  the  use  of  a 
very  fine  emery  paper. 


INSTRUMENTS  FOR  MICROSCOPIC  THIN  SECTIONS.         169 

latter  are  suitable  for  our  purposes.  If  one  has  an  opportunity 
to  get  one  which  has  been  used  for  a  long  time  by  a  barber  and 
has  by  use  acquired  a  perfectly  smooth  and  glistening  surface, 
let  him  take  it ;  it  is  far  better  than  any  that  can  be  had  of  the 
instrument  maker  in  the  most  elegant  case.  But  one  is  com- 
monly referred  to  the  article  which  may  be  had  in  the  market. 
[The  firm  of  J.  R.  Torrey  and  Co.,  of  Worcester,  Mass., 
manufactures  a  razor  strop  which  seems  to  me  to  meet  all  the 
requirements  of  the  phytotomist.  It  is  No.  700  of  their  cata- 
logue "The  combination  hone  and  cushion  belt  strop,"  and  is 
represented  in  Fig.  78.  It  has  four  stropping  surfaces,  sides 
1  and  2  sliding  within  3  and  4,  the  latter  two  being  those 
most  commonly  used  in  keeping  the  razor  sharp.  They  are 
rather  stiff  cushions,  No.  3  having  a  fine  black  surface  for  sharp- 
ening the  edge,  and  No.  4  a  buff,  velvety,  surface  for  giving 
the  final  smoothing  and  polishing  to  it.  Side  No.  1  is  a  fine, 


Italian  rock  hone  which  may  be  used  with  'either  water  or  oil, 
preferably  with  oil,  as  it  is  best  not  to  run  the  risk  of  dampening 
the  wood  and  leather  parts  of  the  strop  by  the  use  of  water. 
It  will  furnish  all  the  sharpening  stone  the  phytotomist  will  need 
for  his  razors.  Side  No.  2  is  a  reddish-brown,  flat,  hard,  leather 
surface,  apparently  provided  with  oxide  of  iron  or  some  such 
like  polishing  paste,  and  is  used  like  any  strop,  first  after  honing 
the  blade.] 

[The  honing  is  done  as  already  described,  though  some  recom- 
mend moving  the  blade  across  the  stone  diagonally  from  point 
to  heel  with  the  edge  foi*ward  instead  of  drawing  it  as  just 
mentioned.  The  stropping  of  the  razor  for  our  work  is  done 
precisely  as  for  ordinary  uses,  laying  it  flat,  edge  and  back 
touching  the  strop,  upon  the  brown,  black  or  buff  surface  as  the 
case  may  be,  and  drawing  it  back  forwards  diagonally  from  heel 
to  point  a  dozen  times  or  more,  up  and  down,  turning  it  upon. 


170 


THE  MICROSCOPE  IN  BOTANY. 


the  back  each  time.  In  finishing  upon  the  buff  surface  it  is  rec- 
ommended to  let  it  lie  not  very  heavily  upon  the  leather,  so  that 
its  soft,  velvety  surface  may  not  tend  to  round  the  edge.  Be- 
fore beginning  the  cutting,  the  author  recommends  drawing  the 
edges  carefully  through  a  bit  of  elder  pith  to  remove  all  adher- 
ing particles  of  grit,  or  steel,  which  may  have  been  left  upon 
it  from  the  sharpening  process.  I  find  that  carefully  drawing 
it  lengthwise  between  the  ball  of  the  thumb  and  first  finger  of 
the  left  hand,  when  pressed  somewhat  closely  together,  will  ef- 
fect the  same  result.  A.  B.  H.] 

For  very  delicate  cuttings,  I  subject 
my  knife,  before  the  removal  of  the 
particles,  to  a  still  further  process, 
in  order  so  to  polish  the  edge  as  to 
remove  the  least  possible  trace  of  every 
kind  of  furrow  which  the  different 
grindings  and  strappings  may  have 
left  upon  it.  This  polishing,  which 
is  commonly  a  failure  with  beginners, 
is  done  in  the  following  manner.  Com- 
mercial chalk  is  sifted  through  a  piece 
of  linen,  and  on  a  thick,  very  smooth 
plate  of  glass  a  little  of  the  sifted  dust 
is  mixed  with  water.  Then  lay  the 
blade  flat,  back  and  edge  upon  the 
glass  and  the  paste,  and  polish  with 
a  circular  motion.  When  the  first  side 
is  finished  take  the  other.  If  I  mistake 
not,  this  process  of  polishing  origi- 
nated with  Hugo  v.  Mohl. 

Now,  that  the  razor  is  perfectly  sharp  the  utmost  care  should 
be  taken  not  to  let  it  get  dull  again.  It  should  never  be  laid 
open  upon  the  table  except  as  the  blade  forms  an  angle  of  about 
270°  with  the  handle.  In  this  position  it  is  impossible  for 
the  edge  of  the  razor  to  come  in  contact  with  the  surface  of 
the  table  on  which  it  lies,  so  as  to  be  damaged.  During  the 
process  of  section-cutting  the  razor  strop  should  never  be 
laid  aside,  but  frequently  used  by  giving  the  knife  several 


FIG. 


INSTRUMENTS  FOR  MICROSCOPIC  THIN  SECTIONS.        171 

strokes  on  it  to  maintain  a  uniform  sharpness.  When  the  work 
is  finished  for  the  time  being,  all  fluids  which  have  been  used 
to  assist  the  cutting  should  be  removed  from  the  knife,  using 
when  needful,  first  water,  then  alcohol  and  ether. 

B.  Scalpels.  Of  scalpel-shaped  knives  the  vegetable  an- 
atomist may  employ,  with  advantage,  the  sorts  illustrated,  natural 
size,  in  Fig.  79.  These  knives  are  not  to  be  employed  for 
making  the  section,  but  for  properly  preparing  the  parts  of  the 
plant  upon  which  the  razor  is  to  be  used. 

The  first  and  second  forms  are  knives  with  straight  blades, 
differing  only  in  respect  to  their  points.  In  the  case  of  I,  the 
point  is  in  the  middle.  This  knife  is  used  to  prepare  pieces  of 
stems,  leaves,  roots  and  other  parts  of  the  plant  held  in  the 
hand,  before  subjecting  them  to  the  razor.  The  knife  II  has 
the  point  lying  laterally  and  forms  an  angle  with  the  edge.  It 
should  be  very  sharp  from  here  to  the  point,  and  is  used  in 
trimming  down  the  section  as  it  lies  on  the  slide  under  the 
mounting  microscope.  Form  number  III,  a  small  knife  with 
slender,  bent  blade  is  convenient  to  use  when  some  peculiar 
form  of  organ  is  to  be  prepared,  parts  of  which  are  difficult  to 
get  at.  It  renders  its  best  service  in  separating  very  small 
flower  buds  when  one  is  studying  the  history  of  the  develop- 
ment of  flowers. 

The  handles  of  these  knives  are  of  ebony,  flat  and  long,  so 
long  that  in  use  they  may  lie  between  the  thumb  and  forefinger, 
say  9,  or  better  11,  cm.  long. 

The  scalpels  are  sharpened  in  the  same  way  as  the  razor.  If 
quite  dull  they  should  be  first  worked  on  the  oil  stone  and  then 
on  the  clay  stone  with  water.  They  should  not  be  turned  over 
right  and  left,  at  every  stroke  as  with  the  razor,  but  sharpen 
one  side  and  then  the  other.  The  final  polish  is  given  on  the 
strop.  It  is  also  recommended  to  keep  a  small  cap  of  elder 
pith  on  the  point  of  the  knives  when  not  in  use  to  protect  them 
from  injury. 

O.  Needles  mid  Lancets.  Needles  of  various  sizes  and 
strength  are  among  the  most  important  requisites  of  the  micros- 
copist,  always,  however,  being  so  strong  that  they  will  not 
spring,  fastened  immovable  in  a  wooden  handle,  Fig.  80,  I,  or 


172 


THE  MICROSCOPE  IN  BOTANY. 


set  iii  a  brass  holder  whose  cap  screws  on  like  that  of  a  lead 
pencil,  Fig.  80,  III.  They  are  useful  in  lifting  the  section 
from  the  razor,  in  spreading  it  out  when  it  is  folded  up  on  the 
slide,  under  the  preparing  microscope,  in  tearing  apart  macer- 
ated plant  tissue,  in  removing  air  bubbles  from  the  mount- 
ing fluid,  and  in  many  other  ways.  For  these  uses  the  needle 
points  should  be  slender  and  sharp. 

Lancets  are  in  the  hands  of  the  phytotomist 
of  far  less  use  than  in  those  of  the  animal 
histologist.  A  form  of  lancet,  which  can 
render  important  service  in  vegetable  histology, 
is  illustrated  in  Fig.  80,  II,  the  lancet-needle. 
It  is  seldom  of  use  for  cutting,  but  in  lifting 
out  large  sections  from  considerable  quantities 
of  fluid  it  is  almost  indispensable.  For  this 
purpose  indeed,  the  forceps  may  be  used,  or  a 
hair  pencil,  or  a  common  needle,  but  by  the 
use  of  the  first  the  section  may  be  easily  in- 
jured and  by  the  last  it.is  apt  to  be  folded  up. 
But  by  the  lancet-needle,  of  the  illustration,  it 
may  be  removed  from  the  fluid  in  the  vessel 
without  injury,  by  putting  the  lozenge-shaped 
surface  under  the  floating  section  and  then 
suddenly  lifting  it  upwards.  Then  the  section 
spreads  out  and  may  be  lifted  up  since  it  has 
more  adhesion  to  the  surface  of  the  lancet  than 
for  the  fluid.  The  use  of  the  lancet-needle  is 
especially  commended  when  dealing  with 
sections  which  have  been  treated  with  reagents 
that  render  them  very  fragile  and  easily  torn 
apart. 

The  needles  are  sharpened  on  the  stone  by 
turning  them  rapidly  with  the  handle,  as  they 
are  being  moved  back  and  forth  over  the  stone, 
dampened  with  oil  or  water. 

Forceps  and  Scissors.  The  forceps,  represented  in  Fig. 
81,  are  useful  in  many  ways.  They  are  best  made  of  steel  with 
long  and  slender  legs  which  are  semicircular  in  section  either 


1        II 

FIG.  80. 

D. 


Ill 


INSTRUMENTS  FOR  MICROSCOPIC  THIN  SECTIONS.         173 


straight  or  a  little  bent.  Their  inner  surfaces  are  cut  like  a  file. 
Forceps  of  brass  or  German  silver  are  useful  but  are  to  be  less 
commended  than  those  of  steel.  They  have  but  one  advantage 
over  those  of  steel,  that  their  dulled  points  inay  be  put  in  order 
again  with  a  file. 

The  scissors  are  a  very  subordinate  instru- 
ment in  the  microscopical  preparation  of  vege- 
table objects.  Small  scissors  with  slender 
straight  or  even  bent  blades  may  be  found  useful 
now  and  then. 

E.  Other  Requisites.  Of  the  other  instru- 
ments which  the  microscopist  frequently  finds 
useful  in  making  preparations,  we  will  name  the 
following. 

a.  Hair  pencils.     Small  hair  or  India  ink 
pencils,  mounted  in    quills  and  provided  with 
long  wood  handles,    are    useful   in   lifting   the 
sections  from  the  razor  blade,   in  removing  su- 
perfluous fluid  from  the  slide,  and  if  perfectly 
dry,  for  brushing  away  dust  from  slides  or  cover 
glasses. 

b.  Glass   rods.      These  are  prepared   from 

longer  glass  rods  or  tubes  about  4  mm.  thick,  by  cutting  off 
any  desired  length,  which  may  be  done  by  softening  them  in  the 
flame  at  a  given  point  and  then  pulling  quickly  apart.  The  ends 
should  be  carefully  rounded  by  melting.  They  may  be  14  to 
20  cm.  long  and  are  chiefly  used  for  carrying  drops  of  the 
reagent  or  mounting  fluid  to  the  slide. 

c.  Porcelain  dishes.     Small,  of  60  to  70  mm.  diameter  and 
about    13  mm.  deep,  with  flat   bottoms  to  stand  secure,  and 
used  to  receive  the  sections,  as  they  are  cut,  in  a  considerable 
quantity   of  water  or   other   fluid    in  order  to  drive  out  the 
air  by  boiling,  or  in  order  to  treat  them  with  reagents  by  heat. 
One   should   be   provided   with  about   half  a  dozen  of  these 
dishes.* 

*  Common  white  individual  butter  plates,  which  may  be  had  cheap  in  any  crockery 
shop,  answer  excellently  for  these  purposes.  Choose  comparatively  thin  and  capacious 
ones,  of  two  different  sizes,  ^ ay  two  dozen  each,  then  one  size  may  be  used  to  cover  over 
the  other  to  exclude  dust  and  retard  evaporation  from  the  preparation.  A.  B.  U. 


FIG.  81. 


174  THE  MICROSCOPE  IN  BOTANY. 

d.  Small  porcelain  crucible  with  cover  of  50  mm.  diameter 
and  35  mm.  high  for  the  maceration  of  vegetable  tissue    (see 
p.  163). 

e.  A  set  of  watch-glasses,    of  different  sizes,  for  example 
of  a   diameter   of  32,    40,    48,    64  and  80  mm.      They  may 
be  applied  to  the  same  uses  as  the  porcelain  dishes,  and  also 
to  find  ing  algae.       A  very  practical    contrivance    for   keeping 
sections  in  many  fluids  protected  from   dust   and   evaporation 

is  represented  in  the  ap- 
paratus pictured  in  Fig. 
82.  It  consists  of  two 
watch-glasses  of  like  size 
with  ground  edges  and 
held  together  with  a  brass 
clamp,  as  shown  in  the 
illustration.  The  under 
watch-glass  contains  the  fluid  with  the  sections. 

[The  "Syracuse  solid  watch-glass,"  shown  in  Fig.  82J,  is 
an  excellent  device,  recently  contrived  by  Dr.  A.  Clifford  Mer- 
cer of  Syracuse,  N.  Y.,  for  use  in  the  histological  laboratory  of 
the  Syracuse  University.  It  is  made  by,  and  can  be  obtained 
from,  the  Syracuse  solid  watch-glass  company.  It  is  a  short 
cylinder  of  glass,  about  55  mm.  wide  and  15  high,  deeply  con- 
cave at  the  top  so  as  to  have  the  internal  form  and  capacity  of 
a  large  watch-glass,  and 
slightly  concave  at  the 
bottom  in  order  to  stand 
firmly  upon  the  table  or 
microscope  stage.  Being 
transparent  and  colorless, 
it  is  well  adapted  to  be 

JtlG.  0-5. 

used  upon  the  micros- 
cope with  transmitted  light,  or  to  be  placed  over  white  or 
black  paper  on  the  table,  according  to  the  background  re- 
quired. As  a  microscopical  bath,  staining  or  dissecting  dish,  it 
possesses  the  well-known  advantages  of  the  watch-glass,  except 
where  heat  is  to  be  applied,  with  the  added  luxury  of  standing 
solidly  on  the  table,  of  immunity  from  breakage,  and  of  fitness 


INSTRUMENTS  FOR  MICROSCOPIC  THIN  SECTIONS.         175 

to  be  piled  upon  each  other  for  economy  of  space  and  as  a  cov- 
ering to  each  other  for  protection  of  their  contents  from  evap- 
oration or  from  dust.  The  glasses  having  only  the  upper  and 
lower  edges  cut  may  be  used  for  many  purposes,  but  those 
having  the  concave  surfaces  also  cut  and  polished  are  preferred 
upon  the  microscope  stage,  especially  for  the  immersion  use  de- 
scribed on  page  32.  R.  H.  W.] 

f.  A  small  wash  bottle,  of  the  form  used  in  chemical  labora- 
tories and  filled  with  distilled  water,  is  of  great  use. 

g.  Spirit  lamp  and  tripod,  or  in  place  of  the  first  a  Bunsen 
burner.  The  tripod  should  be  provided  with  a  brass  netting  of 
narrow  meshes  for  supporting  the  watch-glasses  and  porcelain 
dishes  in  heating  the  sections,  and  for  maceration  with  the  aid 
of  heat. 


FIG.  S3. 


h.  Bell  glasses.  They  are  useful  in  protecting  the  prepar- 
ations from  dust.  For  this  purpose  one  should  have  several  of 
them  from  10  to  30  cm.  in  diameter.  They  should  be  provided 
with  a  knob  on  top  for  convenient  handling.  They  are  used 
simply  by  putting  them  over  the  preparation  as  it  lies  on  the 
table  or  on  paper.  If  it  be  desired  to  keep  the  specimen  moist 
it  may  be  put  on  a  shelf  made  of  zinc,  as  in  Fig.  83,  which 
stands  on  a  plate  filled  with  water.  Over  this  the  proper  bell 
glass  is  put  with  its  edge  immersed  in  the  water. 

i.  A  small  calcium  chlorate  dryer  (of  the  common  labora- 
tory form)  which  can  be  conveniently  employed  in  quickly 
extracting  the  water  from  objects  which  are  to  be  mounted  in 
Canada  balsam.  "  . 

k.  [The  spiral  spring  clip,  scarcely  more  cumbersome  and 
costly  than  the  common  wire  spring  clips  used  for  holding  the 


176 


THE  MICROSCOPE  IN  BOTANY. 


FIG.  84. 


cover  glass  in  position  while  mounting  objects,  is  the  "Nassau  " 
spring  clip  devised  by  Prof.  Libbey  of  Princeton  College  and 
named  after  Nassau  hall  of  that  institution.  In  this  device, 
which  is  made  by  T.  H.  McAllister  of  New  York,  and  shown 
in  Fig.  84,  a  straight  wire  presses  vertically  upon  the  center  of 
the  cover-glass.  The  wire  slides  easily  through 
two  horizontal  folds  of  metal  above,  and  is  held 
down  by  a  spiral  spring  that  encircles  it  and 
presses  against  a  niit  screwed  upon  the  wire  it- 
self. By  twirling  the  upper  end  of  the  wire 
between  the  thumb  and  finger,  any  degree  of 
pressure  may  be  secured,  from  barely  touching 
the  cover-glass  to  a  force  incompatible  with  its 
safety.  After  placing  it  upon  an  object,  the  pres- 
sure may  often  be  changed  to  advantage,  trial  having  shown 
more  or  less  force  to  be  required.  If  considerable  pres- 
sure be  needed  the  bottom  end  of  the  wire  may  be  capped 
with  a  little  block  of  cork  to  prevent  danger  of  cracking  the 
cover-glass ;  but  for  the  lighter  pressures  this  precaution  is  not 
requisite.] 

\The  Parallel    Compressor.      For   examining  thin   objects 
spread  out  to  considerable  size,  or  minute  ones  diffused  sparsely 
through  some  liquid,  it  is  nearly  indispensable  to  have  some 
means  of  lowering  the 
cover-glass   upon  them     /''  *"\% 

gradually,  with  or  with- 
out pressure,  and  with- 
out loss  of  parallelism 
between  the  cover  and 
the  slide.  Some  of  the 
parallel  compressors 
made  for  this  purpose 
have  the  cover-glass  fixed  at  a  certain  height,  and  the  lower 
glass  adjustable  toward  it  by  a  screw  and  spring  motion.  With 
such  arrangement,  the  cover-glass  having  no  upward  move- 
ment is  less  likely  to  be  broken  by  carelessness  when  high 
powers  of  short  focus  are  in  use.  Such  powers,  however, 
cannot  be  advantageously  used  with  carelessness,  in  any  way. 


FIG.  85. 


CUTTING  MICROSCOPICAL  SECTIONS.  177 

For  those  observers  who  find  a  downward  motion  of  the  cover, 
by  screwing  downward,  to  be  more  natural  and  manageable 
than  an  upward  movement  of  the  object  toward  it,  the  Bausch 
and  Lonib  parallel  compressor,  Fig.  85,  offers  a  very  efficient 
instrument.  It  has  a  working  aperture  of  25  mm.,  the  extreme 
size  of  the  cover-glass  being  32  mm.  The  cover  is  carried  down 
by  the  direct  action  of  a  milled  nut  turning  upon  a  screw,  is 
kept  parallel  to  the  plane  of  the  stage  by  two  vertical  pins 
sliding  in  deep  sockets,  and  can  be  turned  aside,  away  from 
the  lower  plate,  by  means  of  a  pivot-motion  of  the  arm  that 
carries  it.  Being  attached  to  the  arm  by  little  steel  spring 
buttons,  it  can  be  replaced  without  delay  if  broken.  R.H.W.J. 
The  foregoing  list  of  instruments  includes  about  all  those  which* 
the  operating  table  of  the  preparator  should  never  lack.  Some 
other  apparatus,  employed  only  on  special  occasions,  as  well  as 
the  contrivances  for  the  preparation  of  permanent  mounts,  will 
be  described  in  their  proper  place. 

IV.     CUTTING  MICROSCOPICAL  SECTIONS. 

Most  vegetable  organs  are  best  adapted  for  cutting  when  fresh. 
Specimens  should  be  immersed  in  water  as  soon  as  they  are  col- 
lected and  likewise  after  having  parts  cut  from  them  for  prep- 
arations. The  sections  should  be  made  with  the  cutting  surface 
covered  with  water  and  the  razor  blade  also  moistened  with  it, 
then  the  sectiwn  may  be  prepared  by  either  of  the  methods 
described  below.  Not  only  are  most  objects  when  fresh  most 
suitable  for  cutting  but  they  are  also  then  in  a  state  most 
favorable  for  the  study  of  the  cell  tissue,  or  the  structure  of 
the  cell  wall,  be  it  of  portions  of  stalk,  leaf  or  flower. 

In  another  group  of  forms  there  are  difficulties  in  the  way  of 
cutting  the  fresh  material.  Many  organs  are  too  soft,  delicate 
and  elastic,  to  offer  sufficient  resistance  to  the  knife  for  section 
making.  Others  consist  of  substances,  partly  hard  and  partly 
soft,  so  that  though  the  former  may  be  cut  well  enough,  the 
latter  will  be  torn.  In  still  other  cases  the  cell  contents  will 
be  displaced  from  their  natural  position  by  cutting,  In  such 
cases  the  substance  must  be  previously  hardened. 
12 


178  THE  MICROSCOPE  IN  BOTANY. 

Materials  for  cutting  may  be  hardened  by  the  use  of  the 
following  media16. 

1.  Alcohol  in   its   anhydrous   state  is   more  suitable   than 
other  fluids,  not  only  for  fixing  the  cell  contents,  but  also  for 
hardening  the  cell  walls.     Hardening  by  means  of  alcohol  is 
indeed  an  old  process.     In  recent  times  it  has  been  employed 
chiefly  by  Strasburger  to  whom  it  has  yielded  the  most  beautiful 
results  in  the  study  of  the  nucleus  and  self-division  of  the  cell. 
The  portion  of  the  plant  to    be  hardened  should  be  put  into 
absolute  alcohol  in  which  the  cell  wall  veiy  soon  becomes  rigid, 
and  the  protoplasm  with  slight  contraction  is  "  fixed."    The  more 
•rapidly  the  fixing  takes  place,  the  more  natural  the  relation  of 
the  part  will  remain.     Many  kinds  of  delicate  tissue  will  not 
bear  the  use  of  absolute  alcohol  on  account  of  the  shrinking  of 
the  membrane  caused  by  the  sudden  withdrawal  of  the  water  by 
the  alcohol.     It  must,   therefore,    be  applied  very  gradually. 
The  plant  must  first  be  put,  for  a  long  time,  in  a  very  weak 
solution,  then  in  a  stronger  and,  finally,  in  absolute  alcohol. 

Again,  in  cutting  other  objects,  we  find  they  behave  most 
satisfactorily  when  they  have  lain  for  a  long  time  in  a  mixture 
of  like  parts  of  alcohol  and  glycerine. 

When  the  object  is  being  cut,  which  has  been  hardened  in  either 
of  these  ways  the  razor  should  be  moistened  with  the  fluids 
used  for  hardening  and  the  cutting  surface  kept  wet  with  them. 

2.  Perosmic  acid.17    A  one  per  cent  solution  of  this  acid  "sets" 
the  protoplasm  by  instantaneous  hardening  still   more  quickly 
than  absolute  alcohol.     In  more  recent  times  it  has  been  em- 
ployed by  many  naturalists  for  this  purpose.    Many  preparations 
which  become  opaque  by  the  use  of  alcohol  retain  their  full  or- 
iginal clearness  by  the  addition  of  one  per  cent  perosmic  acid. 

3.  Chromic  acid  solution  and  Potassium  bichromate.18     The 
former  should  be  a  one  per  cent  solution,  the  latter  in  differ- 
ent degrees  of  dilution,  in  water,  and  it    is  suitable  for   the 

«  Nageli  und  Schwendener,  Das  Mikroskop,  p.  476.—  Sachs  in  Bot.  Zeitg.  1864,  No.  11,  12. 
— Dippel,  Das  Mikroskop,  Bd.  I,  p.  282.— De  Baiy,  Vergl.  Anatom.,  p.  80.— Strasburger,  Be- 
fruchtung  n.  Zelltlieilung,  1878,  p.  38.— Strasburger,  Zellbildung  u.  Zelltlieilung,  1880.  p.  9, 
n.  a.— Poulsen,  Bot  Mikrokerai,  p.  19,/..  deutsche  Uebersetzung,  p.  23 /. 

i?  Frey,  Das  Mikroskop,  p.  103,  /.—Strasburger,  Zellbikl.  u.  Zelltheil.  1880,  p.  39,  172, 
u.  a.— Poulsen  Botanisck  Mikrokemi,  p.  15,/.,  dtsch.  Uebers.  p  18 /. 

18  Hanstein  in  Bot.  Zeitg.,  1868,  p.  697,  ff.  —  Dalmer,  Uebev  die  Leitung  der  PoJlen- 
sehlauche,  p.  18  (Jenaische  Zeitschr.,  Bd.  XIV,  N.  F.  VII,  1880).— Poulsen,  1.  c.,  p.  14,  31, 
dtsch.  Ubeisetz.,  p.  17,  37. 


FREE-HAND  CUTTING. 


179 


hardening  of  many  preparations  which  contain  gums  and  other 
carbohydrates,  resin,  etc.  Resin  for  the  most  part  would  be 
dissolved  in  alcohol,  while  many  kinds  of  gum  by  hardening 
in  alcohol  are  thrown  down  as  a  white  opaque  precipitate.  In 
the  preparation  of  the  potassium  bichromate  .solution  one  may 
use  the  commercial  salt  having  purified  it  by  recrystallization. 

We  now  proceed  to  describe  the  different  kinds  of  micro- 
scopic sections  as  well  as  the  methods  of  their  preparation. 


FIG.  86. 

1.    FREE-HAND  CUTTING-. 

The  razor  should  always  be  held,  when  cutting,  in  the  right 
hand,  in  the  manner  shown  in  Figs.  80  and  87,  the  blade  form- 
ing an  angle  of  112°  to  130°  with  the  handle.  The  hand  grasps 
both  handle  and  blade,  all  five  fingers  being  engaged  in  holding 
the  instrument.  The  thumb  lies  with  its  point  in  the  throat  of 


180  THE  MICROSCOPE  IN  BOTANY. 

the  blade,  the  end  of  the  handle  lying  in  its  first  joint.  The 
index  finger  grasps  the  end  of  the  blade  from  above  with  the 
end  thrown  around  beneath.  The  handle  rests  in  the  inetacar- 
pal  part  of  the  hand  and  is  clasped  by  the  first  and  middle 
phalanges  of  the  middle,  gold,  and  little  fingers.  The  center 
of  gravity  of  the  knife  lies  now  within  the  clasping  hand.  All 
the  fingers  firmly  grasp  the  knife. 

Transverse  Sections.  We  will  now  suppose  we  are  to  pre- 
pare a  section  of  the  stem  of  some  plant.  We  take  the  object, 
which  has  previously  been  given  a  good  cutting  surface  by  means 
of  the  scalpel,  with  the  thumb  and  forefinger  of  the  left  hand 
as  shown  in  Fig.  87,  so  that  the  cut  surface  is  but  a  trifle  above 


FIG.  87. 

the  end  of  the  thumb.  Now  lay  the  knife  flat  on  the  index 
finger  of  the  left  hand,  its  edge  near  the  heel  perpendicular  to 
the  long  axis  of  the  stem.  The  cut  may  now  begin.  If  now 
we  push  the  knife  directly  through  the  plant  like  a  wedge,  we 
shall  find  that  we  have  a  perfectly  worthless  section.  It  will 
be  more  bruised  than  cut.  We  must  cut  with  a  diagonal  motion, 
drawing  the  knife  in  the  direction  of  its  longer  axis,  from  heel 
to  point,  at  the  same  time  that  it  is  pressed  forward  through  the 
stem.*  In  this  way  all  parts  of  the  edge  come  in  contact  with 

*  I  have  to  confess  that  in  my  experience  I  can  do  better  work  by  moving  the  knife  in. 
the  other  direction,  viz.,  from  point  to  heel.  I  suppose  the  reason  is  because,  in  sharpen  ng 
the  knife,  it  is  drawn  from  heel  to  point,  so  that  if  there  are  any  microscopic  lines  along  the 
edge,  or  teeth-like  projections  upon  it,  they  are  placed  in  such  a  position  as  to  take  hold 
better  when  the  knife  moves  through  the  substance  which  is  being  cut,  diagonally,  from 
point  to  heel.  A.  B.  H. 


FREE-HAND  CUTTIXG.  181 

the  section,  which  will  be  found  finally  at  the  upper  end  of  the 
knife  lying  on  the  moist  blade. 

It  should  now  be  lifted  from  the  knife.  This  may  be  done 
by  different  instruments,  the  forceps,  the  hair  pencil,  the  needle 
or  the  lancet-needle.  Only  thick  sections  can  be  handled  with 
the  forceps,  such  as  are  not  injured  by  the  unavoidable  pressure 
involved  in  seizing  them  with  the  forceps,  or  such  as  we  care 
to  examine  only  certain  parts  of  and  so  do  not  mind  the  other 
parts  being  more  or  less  injured  with  the  forceps.  The  hair 
pencil  is  on  many  accounts  to  be  recommended  for  handling 
sections.  In  some  cases  it  is  very  suitable  and  in  others  not, 
since  in  sections  with  large  cells  the  capillary  attraction  which 
attends  its  use  is  quite  likely  to  draw  out  the  cell  contents.  The 
simple  needle  avoids  this,  but  in  lifting  up  sections  with  that, 
large  sections,  at  least,  roll  themselves  up  about  the  needle  and 
so  entangle  themselves  that  it  is  very  difficult  to  put  them  in 
order  again.  This  difficulty  will  be  avoided  by  the  use  of  the 
lancet-needle,  Fig.  80,  II.  When  the  section  lies  'on  the  blade 
of  the  razor  and  a  large  drop  of  water  is  added  it  will  soon 
begin  to  float.  Then  by  putting  the  leaf-like  expansion  of  the 
lancet-needle  under  it  and  lifting  it  quickly  upwards,  the  section 
will  lie  flat  upon  the  needle  and  may  be  immediately  transferred 
to  a  dish  of  water  or  the  slide.  [If  the  section  has  been  cut  in, 
and  is  to  be  transferred  to  water,  a  still  better  way  is  to  hold 
the  razor  blade  inclined  over  the  dish  and  direct  upon  the  sec- 
tiqn  from  the  wash  bottle  a  small  stream  or  a  few  drops  of 
distilled  water  which  will  float  it  off  into  the  dish ;  and  it  often 
happens  too  that  a  section  upon  the  slide,  which  has  got  folded 
or  rolled  up  and  is  too  delicate  to  bear  handling  with  the  needles, 
may  be  perfectly  spread  out  by  directing  upon  it  for  a  few 
seconds  a  fine  stream  of  water  from  a  wash  bottle.  A.  B.  H.] 

Longitudinal  /Sections.  In  making  longitudinal  sections  a 
little  different  method  will  be  employed.  According  to  the 
nature  of  the  material  to  be  cut  the  longitudinal  section  will  be 
made  between  tlie  fingers  or  over  the  finger. 

The  longitudinal  section  between  the  fingers  is  made  in  the 
following  way.  A  small  moistened  piece  of  the  object  to  be 
cut  lengthwise  is  taken  between  the  thumb  and  index  finger  of 


182  THE   MICROSCOPE  IN  BOTANY. 

the  left  hand,  Fig.  88,  or  if  it  is  too  bulky  then  a  prepared 
thin  piece  of  it.  Now,  taking  the  razor,  not  as  before,  but  in 
the  manner  shown  in  Fig.  88,  that  is,  with  the  moistened  blade 
held  exactly  perpendicular,  the  lower  end  of  the  edge  is  placed 
upon  the  object  held  between  the  fingers  in  such  a'way  that  it 
forms  an  angle  of  about  45°  with  the  long  axis  of  the  object. 
The  razor  is  now  drawn  slowly  through  it  between  the  thumb 
and  finger  from  bottom  to  top,  with  the  application  of  a  gentle 
pressure.  Then  we  have  on  the  right  or  left  side  of  the  knife, 
or  clinging  to  the  thumb  or  forefinger,  the  two  longitudinal 
halves  of  the  object.  If  neither  of  these  is  still  delicate  enough 
for  examination  the  operation  must  be  repeated.  Many  people 
can  in  this  way  cut  out  a  longitudinal  section  of  a  very  small 
spherical  object. 


FIG.  88. 

Only  such  objects  are  suited  to  making  a  longitudinal  section 
over  the  finger,  as  are  both  long  and  tough,  as,  for  instance, 
slender  stems,  under-ground  and  aerial  roots.  Take  a  piece 
about  4  to  7  cm.  long  and  making  two  cuts  in  it  near  the  middle, 
about  1  cm.  apart,  half  way  through  it,  and  breaking  it  down 
at  these  places  lay  it  across  the  forefinger  of  the  left  hand,  the 
portion  between  the  cuts  exactly  across  the  back  of  the  finger. 
The  ends  should  be  bent  down  and  held  fast  by  the  thumb  and 
middle  finger.  Now  take  the  razor  as  shown  in  Figs.  86-7, 
and  cut  a  plane  surface  on  the  piece  lying  between  the  two 
cross  cuts,  after  which  very  delicate  sections  may  be  made. 


SECTION-CUTTING  WITH  ELDER  PITH  AND  CORK.         183 


2.     SECTION-CUTTING-  WITH  ELDER  PITH  AND  CORK. 

The  parts  of  many  plants  are  much  too  delicate,  others  too 
small  to  be  held  between  the  bare  fingers  during  the  cutting. 
There  is  indeed  a  whole  series  of  objects  which  the  trained  mi- 
croscopist  can  cut  between  his  fingers  without  injury,  which  the 
beginner  always  crushes  and  only  slowly  learns  to  hold  softly 
in  his  fingers.  But  there  is  still,  even  with  the  most  experi- 
enced, a  considerable  series  of  objects  which  he  must  hold  in 
elder  pith,  or  more  rarely  in  cork,  in  order  to  cut  them.  Be- 
longing to  this  group  are  a  few  objects  with  which  the  writer 
has  had  no  little  experience,  cross  sections  of  the  style  and 
longitudinal  sections  of  the  stigma,  nectary,  ends  of  roots,  the 
younger  and  even  very  youngest  states  of  whole  flower  buds. 
These  are  most  conveniently  held  in  elder  pith.  One  may 
easily  prepare  this  for  himself.  [But  it  is  much  more  con- 
venient, and  if  one's  time  is  worth  anything,  much  cheaper  to 
buy  it  at  the  watchmaker's.  A.  B.  H.]  In  a  large  bush  of 
tiambucus  niyra,  he  will  find  a  large  number  of  already  dead 
and  therefore  dry  branches.  From  these  he  can  easily  obtain 
the  pith  by  splitting  off  the  bark  and  wood  of  the  stem,  in 
pieces  5  to  10  cm.  long  and  8  to  15  mm.  thick.  Since  the  pith 
cylinder  is  somewhat  harder  where  it  joins  the  wood  than  else- 
where, one  can,  in  cases  where  it  is  desirable,  cut  this  away 
with  the  razor  while  it  is  being  used. 

Inclosing  the  object  in  the  elder  pith  is  done  in  the  following 
way.  Taking  a  piece  of  the  pith  it  is  cut  off  even  at  one  end, 
then  split  down,  perpendicularly  to  that  surface,  from  4  to  10 
mm.  with  the  razor.  Now  if  one  has  a  cylindrical  object  to  be 
cut  across  he  drives  a  stout  steel  needle  perpendicularly  into 
the  pith,  in  the  middle  of  the  slit.  Thus  he  makes  a  hole 
which  by  working  the  needle  about  may  easily  be  made  large 
enough  to  take  in  the  object  to  be  cut.  The  pith  is  now  damp- 
ened by  being  plunged  into  water  while  the  object,  which  is 
already  in  that  fluid,  is  taken  and  pushed  into  the  hole  until  it 
sticks  fast.  Since  this  hole,  taking  the  form  of  the  needle,  grows 
somewhat  narrower  towards  the  bottom,  there  is  no  risk  of 


184  THE  MICROSCOPE  IN  BOTANY. 

crushing  or  injuring  the  upper  part  from  which  the  section  is  to 
be  made.  Now  cut  off  the  object  exactly  even  with  the  upper 
surface  of  the  pith  and  again  moistening  both  blade  and  speci- 
men begin  the  cutting  exactly  as  described  above.  The  section 
together  with  the  surrounding  pith  is  transferred  to  a  cup  or 
watch-glass  of  water  and  afterwards  fished  out  for  examination. 

If  a  longitudinal  section  is  to  be  made  the  beginning  of  the 
operation  is  the  same  as  before.  After  that  a  horizontal  notch 
should  be  cut  in  the  top  of  the  pith,  in  the  direction  of  the 
slit,  as  near  as  possible  of  the  size  and  form  of  the  object.  For 
this  purpose  the  scalpel  illustrated  in  Fig.  79,  I,  will  be  found 
useful.  The  specimen  is  then  placed  in  this  receptacle  and  by 
a  gentle  pressure  held  fast.  One  can  by  experience  learn  how 
to  increase  the  pressure  on  the  pith  in  holding  larger  objects, 
for  receiving  which  also  one  may  cut  a  wedge-shaped  piece, 
some  10  mm.  thick,  out  of  the  end  of  the  pith,  instead  of 
making  the  perpendicular  slit. 

Elder  pith  is  almost  always  to  be  preferred  to  cork,  as  well 
on  account  of  its  softness  as  on  account  of  its  uniform  quality. 
All  kinds  of  cork,  even  the  best,  contain  here  and  there  dark, 
hard  concretions  which  make  the  razor  diverge  when  it  strikes 
against  them  in  cutting.  Besides  this  the  cork  has  a  peculiar, 
even  though  but  a  slight,  toughness  which  presents  to  the 
beginner  no  small  difficulty  of  handling.  But  cork  has  some  ad- 
vantages in  cutting  longitudinal  sections  of,  for  instance,  the 
ends  of  roots,  as  suggested  first  by  Nageli,  if  we  mistake  not. 
From  a  large  cork  stopple  make  a  disk  some  10  mm.  thick 
and  on  its  upper  narrow  edge,  by  means  of  a  thick  gum  arabic 
solution  which  dries  quickly,  glue  the  end  of  the  root  to  be 
cut.  Then  bend  over  the  part  of  the  root  which  is  not  fastened 
and  hold  it  down  with  the  thumb  upon  the  cork,  at  the 
same  time  grasping  the  other  side  with  the  index  finger.  It  is 
very  easy  now  to  cut  delicate  longitudinal  sections  of  the  part 
which  has  been  glued  upon  the  cork,  in  the  way  already  de- 
scribed. 

[I  am  inclined  to  believe  that  no  microscopist  ever  uses  cork 
in  section- cutting  except  in  the  way  last  described,  certainly 


SECTION-CUTTING  IN  EMBEDDING  MEDIA.  185 

not  as  a  substance  to  be  cut  through.  It  might,  however,  some- 
times be  used  as  a  backing  to  hold  some  things  up  against  and 
cut  upon.  I  have  found  elder  pith,  on  the  contrary,  almost  in- 
dispensable in  the  free-hand  cutting  of  every  kind  of  the  softer 
vegetable  tissue,  stems  and  leaves  as  well  as  the  most  delicate 
parts  of  flowers.  As  convenient  a  way  as  any  I  have  ever  tried 
in  using  it,  to  cut  transverse  sections,  is  to  split  the  elder  pith 
entirely  through  lengthwise  near  the  middle,  then  carefully 
hollow  out  a  place  on  the  flat  side  of  the  larger  part,  in  the 
middle,  parallel  with  the  longer  axis,  lay  the  specimen  in,  and 
over  it  the  slice  of  pith  which  has  just  been  cut  away,  holding 
the  parts  together  between  the  fingers  while  cutting;  or,  cut  a, 
wedge-shaped  longitudinal  piece  out  of  the  pith,  cut  off  the 
sharp  end  of  the  wedge,  hollow  out  and  enlarge  the  bottom  of 
the  V-shaped  cleft  in  the  pith  as  much  as  is  necessary,  lay  the 
specimen  in  and  replace  the  truncated  wedge.  In  order  to 
guard  against  any  danger  of  injuring  the  delicate  tissue  by  the 
moving  of  the  parts  of  the  pith  upon  each  other,  a  little  rubber 
band  may  be  instantly  sprung  around  it,  or  a  thread  wound 
tightly  about  it  and  tied,  or,  as  mentioned  on  page  167,  one  may 
hold  the  elder  pith  with  the  specimen  in  it,  in  a  hand-vise. 
Then  the  cutting  may  proceed  at  leisure,  and  one  may  lay  the 
specimen  down  to  get  the  use  of  his  left  hand  in  manipulating 
the  section,  already  cut,  without  fear  of  deranging  it.  It  is 
clear  also  that  specimens  for  longitudinal  sections,  of  plant  parts 
not  longer  than  the  diameter  of  the  pith  cylinder,  may  be  fixed 
in  the  same  way  by  cutting  out  the  bed  for  the  specimen 
transversely  across  the  flat  side  of  the  semi-cylinder.  The  pith 
can  then  be  cut  as  before,  transversely,  at  least  as  far  as  to  the 
middle  of  the  specimen.  A.  B.  H.] 


3.     SECTION-CUTTING  IN  EMBEDDING  MEDIA. 

A  number  of  things  which  are  to  be  cut  are  so  small  as  to  be 
almost  or  quite  invisible  to  the  naked  eye.  These  should  be 
previously  mixed  in  a  considerable  quantity  in  a  hardening  me- 
dium and  then  by  making  random  cuts  through  this  the  small 


186  THE   MICROSCOPE   IN  BOTANY. 

objects  will  be  cut  into  fine  layers.  Botanical  objects  of  this 
kind  are  such  things  as  starch  grains,  pollen  grains,  spores,  and 
delicate  leaves  of  mosses  and  liverworts.  Making  sections  of 
such  embedded  substances  is  done  in  the  following  way.  To  a 
thick  solution  of  gum  arable  add  some  concentrated  glycerine.* 
Put  a  drop  of  it  in  a  watch-glass  and  knead  up  with  it,  into  a 
tough  dough,  a  quantity  of  starch  grains.  Put  this  in  several 
layers  and  in  sufficient  quantity  upon  the  end  of  a  cork  stop- 
per and  leave  it  to  harden.  When  the  mass  has  become  suffi- 
ciently hardened  to  be  cut  with  the  razor,  the  cutting  should 
be  done.  Then  put  it  in  water  and  in  a  few  moments  the 
gum  will  dissolve  away  and  the  dissected  grains  will  float 
out  free.19 

From  several  sides  lately  there  have  come  commendations  of 
glycerine  jelly  as  an  embedding  medium.  According  to  some 
experiments  of  my  own  in  this  matter  1  believe  that  for  many 
botanical  purposes  it  may  really  be  commended  although  in  order 
to  give  a  definite  judgment,  these  experiments  must  be  carried 
further  in  the  future.  In  investigations  into  the  history  of  the 
development  of  flowers  glycerine  jolly  may  hereafter  be  found 
a  medium  of  extensive  application.  It  is  frequently  very  de- 
sirable in  this  investigation  to  keep  the  parts  of  the  flower, 
.where  for  instance  a  section  of  a  very  young  bud  has  been  made, 
in  their  relative  position  when  they  are  put  under  the  micro- 
scope. This  is  very  difficult  to  do  when  the  cutting  has  been 
done  with  water  or  alcohol.  But  this  is  often  accomplished 
where  the  object  has  been  impregnated  with  gelatine  previous 
to  cutting.  It  should  be  put  first  of  all  in  the  gelatine  and  then 
by  a  process  to  be  explained  below,  freed  from  air  and  the  gela- 
tine forced  into  the  spaces  between  the  floral  parts.  The  pro- 
cess, however,  is  somewhat  detailed. 

Koch20  has  given  a  process  for  embedding  small  and  delicate 
parts  of  plants  in  a  mixture  of  tallo\y  and  paraffins  in  order  to 

*  Probably  the  well  known  Farrant's  Medium  would  answer  this  purpose  sufficiently 
well.  It  is  made  as  follows.  Dissolve  4  parts  by  weight  of  picked  gum  arable  in  4  parts  cold 
distilled  water  and  add  2  parts  strong  glycerine,  strain  through  linen  and  keep  in  glass 
stoppered  bottles  with  a  little  camphor  gum.  A.  B.  H. 

19  Hartig,  Entwicklungsgeschichte  des  Pflanzenkeimes,  p.  87. 

20  Koch,  Untersuchungen  Ueber  die  Entwicklung  der  Cuscuteen,  1874  (Botanische  Ab- 
handl.  herausgegen,  v.  Hanstein,  Bd.  II,  Holt  3,  p.  24). 


CUTTING  SECTIONS  WITH  A  MICROTOME.  187 

cut  them  into  sections,  a  process  which  has  been  in  manifold  use 
in  zoology  for  a  long  time. 

He  says:  "The  mixture  of  like  parts  of  tallow  and  paraffine 
has  so  low  a  melting  point  and  stiffens  so  rapidly,  that  the  plant 
laid  on  it  loses  scarcely  any  water  and  therefore  suffers  no  ap- 
preciable shrinking.  Before  putting  the  specimen  into  the 
mixture,  it  is  well  to  dip  it  for  a  minute  or  less  into  alcohol  and 
then  let  the  alcohol  evaporate.  The  purpose  of  this  is  to  remove 
any  water  that  may  be  attached  to  the  outside  and  allows  the 
melted  medium  to  take  fast  hold  of  the  specimen.  If  this  is 
neglected,  bubbles  and  spaces  will  be  formed  about  the  specimen 
which  will  make  it  go  badly  with  the  section-cutting.  A  quan- 
tity of  the  melted  medium  is  put  upon  a  slide  and  the  specimen 
immersed  in  it.  After  some  minutes  it  will  become  sufficiently 
solid  to  proceed  with  the  cutting. 

For  the  removal  of  the  fat  from  the  section,  they  should  be 
first  placed  in  benzole  and  then  in  alcohol,  and  at  last  treated 
as  fresh  sections  with  reagents. 

An  essential  requirement  in  the  application  of  this  method  is 
that  the  embedding  medium  shall  have  a  consistency  propor- 
tionate to  the  nature  of  the  material  to  be  cut.  This  may  be 
attained  by  changing  the  relative  proportions  of  the  tallow  and 
the  paraffine.  While  like  parts  of  each  make  a  medium  suited 
to  the  harder  parts  of  plants,  the  softer  parts  require  a  mixture 
of  one  part  of  paraffine  to  two  of  tallow.  This  proportion  may 
be  carried  still  further. 

Like  results  are  reached  when  very  delicate  objects  are  treated 
if  they  are  cut  before  the  mixture  is  quite  hardened.  Finally, 
if  fresh  plants  are  to  be  cut  they  should  be  immersed  in  alcohol 
for  at  least  a  day.  But  these  cut  but  poorly  at  best  in  this  way." 


[4.    CUTTING-  SECTIONS  WITH  A  MICROTOME.] 

[Our  author  has  given  in  the  text  no  account  of  wrhat  Amer- 
ican microscopists  rind  to  be  an  almost,  and  sometimes  quite, 
indispensable  aid  to  their  investigations,  viz.,  a  good  section- 
instrument,  or  microtome.] 


188  THE   MICROSCOPE  IN  BOTANY. 

[The  Common  Section  Machine.  The  more  common  forms 
are  represented  in  Fig.  89.  The  material  to  be  cut  is  put  into 
the  well  and  either  embelded  in  paraffiue  or  otlur  like  sub- 
stance, or  packed  close  and  firm  with  elder  pith,  and  the  cutting 
is  done  with  a  very  sharp,  straight-edged  knife,  Fig.  92,  mov- 
ing directly  forward  or  diagonally  upon  the  even  surface  of 
the  metallic  top.  The  material  is  raised  in  the  well  after  each 
stroke  of  the  knife,  so  as  to  cut  a  section  of  any  desired  thick- 
ness, by  turning  the  large  milled  and  graduated  screw  head 
at  the  bottom,  which  moves  a  piston  in  the  well,  upon  which 
the  material  rests.  A  is  provided  with  a  set  screw  at  the  side 
designed  to  turn  in  and  fix  the  specimens  firmly  against  the 


Jf'iu.  89/1.  Jb'IG.  89B. 

Wall  of  the  well.  This  is  more  particularly  designed  for  cut- 
ting wood  sections  which  would  need  no  other  means  of  keeping 
the  material  in  place.  B  is  intended  for  cutting  larger  sections 
of  softer  embedded  tissue.  It  has  a  larger  well  and  is  provided 
with  a  glass  top.  Both  have  strong  clamps  by  which  to  fasten 
them  to  the  table  when  being  used.  A  razor  instead  of  the  knife 
might  sometimes  be  used  in  cutting  soft  tissue  with  B  provided 
the  razor  have  a  very  stiff  blade,  and  a  veiy  straight  edge.] 

[The  Providence  Microtome.  So  called  from  being  the  joint 
invention  of  several  gentlemen  of  Providence,  E.  I.  The  orig- 
inal form  was  first  designed  by  Mr.  IS".  N.  Mason  of  that  city, 


THE  PROVIDENCE  MICROTOME. 


189 


who  has  used  it  for  several  years  in  zoological  work.  The  in- 
strument was  perfected  by  Rev.  J.  D.  King,  Microscopist  of  the 
Martha's  Vineyard  Summer  Institute,  one  of  the  gentlemen  re- 
ferred to,  by  whom  it  is  manufactured  and  sold.*  In  its  present 
form  it  is  perhaps  equalled  by  no  microtome  made,  for  extreme 
precision  of  movement  and  consequent  accuracy  of  performance 
in  cutting  sections.  With  a  good  knife  in  good  order,  sections 
of  10  iJ.  to  25  /.*  thick  can  be  made  without  difficulty,  and  all 
alike.  It  consists,  as  is  seen  in  Fig.  90,  of  a  heavy  iron  bed,  J9, 
a  knife  carrier,  A,  and  the  usual  apparatus  for  holding  and 
moving  the  object  to  be  cut,  gj.  The  iron  bed  which  furnishes 
the  clamp  k,  and  a  solid  support  for  the  knife  carrier  and  object 


FIG.  90. 


holder  is  13.8  cm.  long,  and  5.7  cm.  wide,  6.8  cm.  deep.  Ce- 
mented to  its  top  is  a  brass  plate,  A,  6.5  mm.  thick.  Rising 
through  and  above  this  is  the  cylindrical  tube  or  object  holder,  j, 
29  mm.  in  diameter.  It  projects  10  mm.  above  the  surface  of 
the  brass  plate  and  to  within  0.5  mm.  of  the  upper  surface  of 
the  knife  carrier.  It  has  an  inner  cylindrical  piston  15  mm.  in 
diameter  and  a  sleeve  around  this  which  may  be  used  with  the 
piston,  when  it  is  desired  to  have  a  larger  well,  having  a  di- 
ameter of  19  mm.  On  each  side  of  the  brass  plate  and  rising 
1  mm.  above  its  upper  surface  is  an  iron  bar  7  mm.  thick  rim- 

*  Address  Rev.  J.  D.  King,  Cottage  City,  Mass. 


190  THE  MICROSCOPE  IN   BOTANY. 

ning  the  whole  length  of  the  bed  and  screwed  fast  to  it.  These 
are  the  ways  or  tracks  upon  which  the  knife  carrier  slides.  The 
knife  carrier  consists  of  a  solid  plate  of  hrass  13  cm.  long  and 
8.6  cm.  broad,  8  mm.  thick,  with  projections  along  both  sides 
6  mm.  thick  and  13  mm.  deep,  which  fit  down  over  the  outside 
of  the  iron  ways  just  mentioned.  The  inside  of  these  projec- 
tions and  the  adjoining  under  surfaces  of  the  brass  plate  are 
planed  and  polished  so  as  exactly  to  fit  over  and  upon  the  smooth 
iron  tracks  in  such  a  way  that  the  carrier  moves  freely  but  with 
the  utmost  precision  back  and  forth  upon  them.] 

[The  brass  plate,  -4,  has  an  oblong  opening  cut  in  its  middle 
9.6  cm.  long  and  3.3  cm.  wide,  through  which,  when  in  place, 
the  cylindrical  object  holder  projects,  very  nearly  to  the  upper 
surface  of  the  plate.  The  plate  is  provided  along  its  sides  and 
ends  with  a  series  of  screw  holes,  to  receive  the  milled  head 
screws,  act,  of  the  clamps,  6cZ,  by  means  of  which  the  knife,  e, 
is  made  fast  to  the  carrier,  and  may  be  set  at  any  desired  obliq- 
uity to  the  line  of  motion  of  the  carrier.  The  knife  has  a  heavy 
strong  plano-concave  blade  with  a  straight  edge,  and  is  laid  flat 
upon  the  carrier  and  securely  clamped  down  at  heel  and  point. 
It,  therefore,  will  not  spring  in  the  least  and  may  be  depended 
on  to  do  work  of  very  great  precision.  It  is  used  for  cutting 
all  kinds  of  wood  sections,  and  such  other  tissue  as  can  be  cut 
by  simply  packing  in  elder  pith  or  embedding  in  paraffine.  The 
method  of  embedding  in  paraffine  mixtures  for  which  I  am  in- 
debted to  Rev.  Mr.  King  is  as  follows  :] 

[Screw  up  the  plug  to  within  2  mm.  of  the  top,  and  fill  with 
vaseline  made  as  hard  as  can  be  used  with  paraffine.  Stick  the 
end  of  the  object  to  be  cut  in  the  vaseline,  giving  the  object  the 
desired  position.  Then  screw  down  till  the  upper  end  of 
the  object  is  below  the  surface  of  the  plate,  and  pour  in  a  small 
quantity  of  melted  paraffine  —  too  much  will  melt  the  vaseline 
and  displace  the  object. — When  this  is  hardened,  fill  the  well 
with  paraffine.  The  paraffine  should  be  softened  with  vaseline  or, 
better,  paraffine  wax.  No  rule  for  proportions  can  be  given,  as 
the  consistency  of  the  embedding  medium  must  depend  upon  the 
hardness  of  the  object  to  be  cut.  It  should  be  harder  in  the 
summer  than  in  the  winter.  The  temperature  too  must  be  de- 


THE  TAYLOR  FREEZING  MICROTOME.  191 

termined  by  the  experience  and  judgment  of  the  operator.  If 
the  paraffine  is  too  hot  it  will  boil  up  in  the  well  and  will  not 
cool  solid,  if  too  cool  it  will  not  properly  fill  in  around  the  ob- 
ject. If  the  object  is  very  porous  it  will  be  well  to  give  it  a 
coating  of  mucilage  and  let  it  dry  before  embedding  in  paraffine 
as  this  will  keep  the  paraffine  from  filling  the  pores. 

When  all  is  complete  in  the  well,  the  paraffine  may  shrink. 
In  that  case,  make  a  very  thin  plug  of  wood  and  drive  it  down 
by  the  side  of  the  paraffine  quite  to  the  bottom  of  the  well. 
When  the  section  is  cut,  put  it  in  distilled  water,  when  the  sec- 
tion will  drop  out  clean  from  the  paraffine.  But  if  this  fails  soak 
in  strong  alcohol.] 

[  The  raylor  Freezing  Microtome.  As  has  already  been  in- 
dicated in  the  author's  account  of  free-hand  cutting,  there  are 
many  vegetable  tissues  of  too  soft  or  too  delicate  a  nature  to 
furnish  sufficient  resistance  to  the  knife  to  be  cut  into  sections. 
He,  therefore,  suggests  certain  means  for  hardening  the  tissue. 
But  these  hardening  processes  take  time  and  care  and  are  at 
best  not  very  satisfactory  or  successful.  The  zo  Jlogist  has 
long  ago  met  the  same  difficulty  in  dealing  with  animal  sub- 
stances, and  has  contrived  various  means  of  overcoming  it.  For 
some  time  past  he  has  been  employing  artificial  refrigeration  as 
among  the  best  and  most  expeditious  means  of  hardening  tis- 
sue. Any  tissue,  no  matter  how  loose,  or  soft  or  delicate  in  its 
normal  state,  if  permeated  with  aqueous  fluids,  can  be  frozen 
solid  and  cut  with  a  razor  or  in  a  microtome  like  any  other  solid 
substance.] 

[So  far  as  I  know,  botanists  have  made  very  little  use  of  this 
extremely  simple  and  effective  method  of  temporarily  harden- 
ing, for  purposes  of  section-cutting,  the  soft  and  delicate  tissue 
with  which  their  studies  are  often  concerned.  And  yet  it  is 
susceptible  of  a  far  wider  and  readier  application  to  the  needs 
.and  uses  of  the  vegetable,  than  to  those  of  the  animal  histologist ; 
for,  all  fresh  vegetable  tissues  are  in  nature  completely  perme- 
ated with  aqueous  fluids.  They,  therefore,  require  no  previous 
preparation  to  make  them  ready  for  the  freezing  process,  and 
parts  of  dried  plants  only  need  soaking  in  water  till  they  are 
permeated  and  softened  by  it ;  and  vegetable  tissue  so  far  as 


192  THE   MICROSCOPE  IN  BOTANY". 

I  can  discover  is  in  no  way  injured,  or  in  the  least  degree 
changed  by  the  freezing.] 

[The  process  is  almost  equally  applicable  to  all  kinds  of  soft 
vegetable  substances,  the  leaves  and  stems  of  the  higher  plants, 
parts  of  buds  and  flowers,  ovaries  and  ovules  in  every  stage  of 
development,  soft  portions  of  roots,  tubers  and  root  stalks,  the 
flesh  or  rind  of  vegetables,  fruits,  and  soft  seeds,  pollen  grains, 
spores  of  cryptogams,  fronds  of  algae,  lichens,  hepaticae,  ferns, 
fungi,  etc.] 

[Various  means  have  been  proposed  and  adopted  for' produc- 
ing artificial  refrigeration,  as,  for  example,  chemical  mixtures  ; 
the  rapid  evaporation  of  volatile  liquids  such  as  ether  and  rhigo- 
line;  mixtures  of  alcohol  and  ice,*  salt  and  ice,  etc.  Several 
different  microtomes  have  been  devised  especially  for  utilizing 
one  or  the  other  of  these  methods  of  freezing.  By  far  the  sim- 
plest and  most  convenient  of  them  is  the  Taylor  freezing  micro- 
tome, represented  in  Fig.  91.  It  is  the  invention  of  Dr. 
Thomas  Taylor,  Microscopist  of  the  Department  of  Agricul- 
ture, Washington,  D.  C.] 

\_A  is  a  revolving  plane  of  glass  and  brass  cemented  together, 
about  10  cm.  in  diameter,  and  securely  fastened  to  a  short 
cylinder,  5cm.  in  diameter,  which  screws  upon  another  cylin- 
der which  in  its  turn  is  made  fast  to  the  wooden  base.  Inside 
the  cylinder  upon  which  the  plane  revolves  and  isolated  from  it 
is  a  cylindrical  brass  chamber  3.9  cm.  in  diameter,  fastened 
to  the  wooden  base  and  entered  by  two  metallic  tubes,  the 
larger  one  connected  by  the  rubber  tube  t,  with  the  pail  «, 
placed  upon  a  convenient  bracket  or  other  support  above  the 
level  of  the  microtome.  To  the  other  tube  is  joined  the  rubber 
tube,  tb,  which  discharges  the  cold  liquid  into  the  pail  placed 
beneath  it.] 

[The  edge  of  the  revolving  plane,  A,  is  graduated  and  the 
movable  pointer,  e,  indicates  how  far  the  plane  has  been  re- 
volved in  a  given  instance.  The  screw  thread  of  the  cylinder 
measures  about  0.634  mm.  So  that  if  the  plane  is  revolved  -±  of 
its  circumference  it  would  be  raised  or  lowered  as  the  case  might 
be,  0.0634  mm. ;  or  L  would  elevate  or  depress  it  0.0317  mm.  ; 

*Prof.  S.  H.  Gage  in  Science  Record,  Apr.  15, 1884,  p.  134. 


THE  TAYLOR  FREEZING  MICROTOME. 


193 


1,  0.01268  mm.  ;  JL,  0.00634  mm.  Thus  by  a  simple  calculation 
it  is  easy  to  determine  how  far  to  turn  it  to  get  any  required 
perpendicular  motion,  answering  to  the  desired  thickness  of  the 
section.] 

[The  freezing  is  accomplished  by  thoroughly  mixing  coarse 
salt  with  snow  or  finely  pulverized  ice  in  the  upper  pail.     Pour 

on  a  little  water  to  start  the  flow  of  the 
freezing   liquid.      When   the   metallic 
chamber,  which   is    immediately   filled 
with  a  liquid  at  a  temperature  of  about 
—  18°  C.  becomes   frosted    over  with 
the  moisture  condensed  from  the  atmos- 
phere, the  instrument  is  ready  for  work. 
The  flow  of  the  cold  brine  may  be  best 
regulated  by  u^ing 
a  faucet  or  stop- 
took  with  the  pail 
a,  by  which  any 
desired    qunntity 
of  water  may  be 


permitted  to  pass.  The  water  should  be 
poured  back  from  the  lower  pail  as  it  runs 
low  in  the  upper  one.  This  may  be  re- 
peated as  long  as  it  remains  below  the 
freezing  point.  A  vessel  holding  10  or  12 
litres  full  of  snow  and  salt  with  perhaps 
J  a  litre  of  water  poured  on  at  the  start 
will  furnish  refrigeration  sufficient  for  four  or  five  hours'  steady 
work.] 

[The  method  of  using  this  microtome  is  extremely  simple. 
Keep   on   hand  a  thick  syrupy  solution  of  pure  guni  arabic, 

13 


FIG.  91. 


194  THE  MICROSCOPE  IN  BOTANY. 

which  sifter  dissolving  has  been  strained  through  a  linen  cloth, 
and  to  which  has  been  added  a  little  carbolic  acid  to  prevent  the 
growth  of  fungi.  Put  a  drop  or  two  of  this  solution  on  the  top 
of  the  freezing  chamber  and  immediately  immerse  in  it  the  tis- 
sue to  be  cut,  which  has  been  previously  moistened  with  water, 
being  careful  to  cover  it  well  with  the  gum  solution.  In  two 
or  three  minutes  the  whole  will  be  frozen  into  a  grayish,  solid, 
even  crystalline-appearing  mass,  and  the  cutting  may  now  begin. 
Now  turn  the  revolving  plate  backwards  till  it  is  brought  nearly 
to  a  level  with  the  top  of  the  frozen  mass  upon  the  cylinder. 
Then  moisten  the  glass  surface  liberally  with  water,  and  hold- 
ing the  knife  upon  it  as  directed,  with  a  straight  motion,  cut 
away  the  top  of  the  mass  and  make  it  ready  for  taking  off  a 
section.  Now  turn  the  revolving  plane  forward  the  right  dis- 
tance to  lower  it  the  desired  thickness  of  the  section,  and  with 
the  knife  make  a  diagonal  or  directly  forward,  steady,  even,  but 
not  too  quick  cut.  Remove  the  section  from  the  knife  with  a  hair 
pencil  dipped  in  water,  into  a  dish  of  water  provided  for  it. 
Or  if  the  section  is  a  small  one  of  tissue  not  too  delicate  five,  ten, 
or  more  may  be  made  and  allowed  to  accumulate  upon  the  knife 
before  bein£  removed.] 

[The  advantages  of  this  process  of  hardening  and  cutting 
vegetable  tissues  are  so  manifest  as  hardly  to  require  mention. 
No  other  method  of  hardening  leaves  the  tissue  and  cell  contents 
unchanged,  and  no  free-hand  cutting  can  make  the  sections  of 
so  uniform  thickness,  or  determine  with  any  approach  to  exact- 
ness what  the  thickness  shall  be.  By  this  method,  which  com- 
bines in  one  act  the  embedding  and  hardening,  the  section  can 
be  made  in  any  direction  across  the  specimen  at  will,  transverse, 
tangential,  radial  or  diagonal ;  and,  if  the  object  is  not  too  large, 
separate  portions  may  be  so  placed  on  the  freezing  chamber  as 
to  allow  of  all  these  different  sections  being  made  at  the  same 
time  by  one  stroke  of  the  knife.] 

[In  order  to  secure  a  somewhat  greater  precision  in  the  ad- 
justment for  thickness  of  section,  I  have  made  some  changes  in 
my  own  instrument,  changes  which,  however,  in  no  way  affect 
the  nature  or  value  of  the  extremely  simple  and  effective  refriger- 


CUTTING  SECTIONS  WITH  A  MICROTOME.  195 

ating  apparatus.  I  make  the  glass  plate  fast  and  the  freezing 
chamber  movable.  Turning  the  glass  plate  down  to  the  limit  of 
the  screw  it  is  tightened  up  and  left  permanently  in  that  position. 
By  fastening  a  ring  of  hard  wood  msjle,  the  outer  cylinder, 
I  furnish  a  support  for  the  freezing  chamber  which  holds  it 
firmly  in  the  center  of  the  cylinder,  but  permits  it  to  move  freely 
up  and  down.  Beneath  all,  the  wooden  base  is  cut  away  so  as 


FIG.  92. 

to  give  the  freezing  chamber,  its  support,  pipes,  etc.,  space  for 
moving  about  15  mm.  The  chamber  and  its  attachments  are 

W 

made  fast  at  the  bottom  to  a  small  brass  cylinder  about  4  cm. 
high  and  18  mm.  in  diameter,  into  the  center  of  which  from 
below  works  a  micrometer  screw  with  a  thread  1.058  mm.  wide. 
This  has  a  large  graduated  head,  by  turning  which  the  freezing 
chamber  with  its  section  material  maybe  raised  any  desired 
distance  .from  5  /*  upwards  and  the  section  made.*] 

[  The  Section  Knife.  The  knife  used  with  these  instruments 
is  the  common,  straight-edged  section  knife  sold  by  the  op- 
ticians, and  illustrated  in  Fig.  92.  Two  things  are  to  be  re- 
quired of  it.  The  one  is  that  the  edge  be  as  nearly  absolutely 
straight  as  it  is  possible  to  make  it,  and  the  other  is  that  it 
should  be  of  such  quality  and  temper  of  steel  as  that  it  will 
take  and  hold  the  finest,  smoothest  and  keenest  edge  possible. 
In  using,  it  should  be  held  at  perhaps  an  angle  of  30°  to  the 
horizontal  plane  of  glass  over  which  it  moves.  A.  B.  H.] 

*T\vo  microtomes  recently  introduced  into  this  country,  the  "Thoma  Sliding  Micro- 
tome" from  Germany,  and  tiie  "  C  a  Id  well  Automatic  Microtome"  from  England,  are  bein* 
used  with  much  favor  in  some  of  our  zoological  laboratories.  They  are  chiefly  valuable 
for  the  extreme  tenuity  of  the  sections  which  they  are  capable  of  cutting  from  the  compar- 
atively delicate  material  of  the  animal  organism,  and  for  the  special  contrivance  in  the 
"Caldwell"  form  for  cutting  -'ribbons"  of  consecutive  sections.  But  for  work  UMOU  the  more 
difficult  material  of  the  botanical  laboratory  I  do  not  consider  them  as  practically  useful 
as  either  of  the  t\vo  desciibed  above,  while  neither  of  them  has  the  freezing  apparatus, 
which  makes  the  "  Taylor  Microtome"  so  valuable  in  dealing  with  the  soft,  water-holding 
tissue  of  many  fresh  vegetable  organisms;  besides,  the  foreign  are  much  more  expensive 
than  the  American  instruments.  A.  B.  H. 


196  THE  MICROSCOPE  IN  BOTANY. 


V.    FURTHER  TREATMENT  OF  THE  SECTION. 

Having  made  the  sections  and  immersed  them  in  water,  we 
will  now  undertake  to  show  how  they  are  to  be  further  treated 
before  they  are  put  under  the  microscope  for  examination.  [A 
little  instrument  shown  in  Fig.  93  will  be  found  very  conven- 


FIG.  93. 


ient  in  lifting  sections  out  of  the  water  and  indeed  in  transfer- 
ring them  from  any  fluid  to  another,  or  to  the  slide.  It  is  a  little 
trowel  or  lifter,  metallic  and  nickel  plated.  A.  B.  IL] 


A.    REMOVING  THE  AIB. 

We  shall  observe  that  in  many  sections  the  cellular  tissue  is 
partly  filled  with  small  air  bubbles.  Cells  filled  with  air  show 
a  thick  black  outline  on  the  inner  wall  which  prevents  the  nat- 
ural structural  relations  of  the  wall  from  being  fully  made  out 
if  at  all.  It  is  therefore  first  of  all  necessary  to  remove  these 
minute  bubbles  from  the  tissue.  It  may  be  done  in  the  following 
ways,  sometimes  best  in  one  and  sometimes  in  another. 

(a)  Boil  distilled  water  in  a  porcelain  cup,  let  it  cool  and 
then  put  the  sections  into  it.  The  air  will  soon  be  absorbed  in 
the  deaerated  water  and  after  a  little  time  will  disappear  en- 
tirely. This  is  in  most  cases  an  infallible  means  —  only  re- 
membering to  always  use  freshly  boiled  water. 

(6)  The  air  will  be  drawn  out  more  forcibly  by  putting  the 
sections  in  distilled  water  and  then  bringing  the  water  to  the 
boiling  temperature,  and  afterwards  allowing  it  to  cool.  Deli- 
cate specimens,  and  those  whose  cell  contents  are  to  be  studied 
will  not  bear  the  heat. 

(c)  Sometimes  the  air  disappears  when  the  section  is  re- 
moved from  the  water  into  absolute  alcohol  for  a  considerable 


SECTION  UNDER  THE  PREPARING  MICROSCOPE. 


197 


time  and  then  returned  to  the  distilled  water.     This  method 
was  first  suggested  by  Schacht. 

(d)  The  air  may  be  removed  by  means  of  an  air  pump. 
For  this  purpose  many,  and,  for  the  most  part,  very  expensive 
contrivances  have  been  proposed.  But,  as  according  _ 
to  our  opinion,  he  is  the  best  experimenter  who  does 
his  work  with  the  simplest  possible  apparatus,  we 
will  describe  here  a  very  simple  but  serviceable  con- 
trivance, which  one  can  easily  make  for  himself. 
Fig.  94  represents  a  thick  walled  glass  tube  a  a  of 
about  22  mm.  interior  diameter  and  15  to  18  cm. 
long,  with  one  end  closed.  A  small  piston  of  strong 
zinc  plate  is  fitted  to  this  by  winding  with  tow  or  other 
packing,  and  has  a  small  hole  in  the  middle  covered 
with  an  air-tight  clapper  valve  v.  The  piston  has  a 
handle  k.  Into  the  cylinder  is  put  a  little  boiled 
water  with  the  air-containing  section  c.  The  piston  is 
now  driven  down  to  the  level  of  the  water  and  again 
drawn  up.  The  valve  closes,  the  space  beneath  be- 
comes almost  a  perfect  vacuum,  and  into  this  the  air 
/.  .  .,  (.  /»  •  FIG.  94. 

from  the  section  streams  up  in  the  torm  ot  minute 

bubbles.     The  contents  of  the  tube  may  now  be  poured  out  into 
a  cup  and  the  section  removed  to  the  slide. 


B.     HANDLING  THE  SECTION  TJNDEB,  THE 
MICROSCOPE. 


PREPAKING- 


The  section  having  been  carefully  freed  from  adhering  air, 
it  is  placed  in  a  drop  of  fluid,  water,  glycerine,  etc.,  according 
to  circumstances,  on  a  slide.  It  will  often  be  found  impossible 
to  prevent  the  edges  from  being  folded  under  or  OA^er.  The 
preparing  microscope  is  used  to  properly  spread  it  out  for  ex- 
amination, which  is  not  by  any  means  a  difficult  thing  to  do. 
Clamp  the  slide  to  the  stage  in  such  a  position  that  the  object 
will  come  over  the  middle  of  the  opening  in  it  and  then  adjust 
the  mirror  so  as  to  throw  up  a  beam  of  light.  Adjust  the  lens 
and  examine  it  to  see  if  it  is  all  right,  and  if  not  how  it  is  wrong. 
By  focussing  up  and  down  it  will  be  easy  to  tell  what  parts  are 


198  THE   MICROSCOPE  IN  BOTANY. 

folded  over  and  what  under,  if  any.  Then  take  a  needle  in  each 
hand  and  with  that  in  the  left  hand  hold  the  section  down  gently 
and  with  the  other  arrange  the  folded  parts.  If  only  a  part  of 
the  section  is  to  he  examined  and  the  others  are  folded  over  it, 
remove  them  entirely  from  the  slide.  This  may  he  done  by 
means  of  the  lancet-needle,  or  the  small  scalpel,  or  even  the 
scissors,  holding  down  the  section  with  the  needle  as  before. 
Having  now  rightly  laid  out  the  section  to  our  satisfaction,  we 
should  put  a  cover-glass  over  it.  The  section  can  now  be  trans- 
ferred to  the  compound  microscope  to  be  studied.  If  this  can- 
not be  done  at  once  it  may  be  kept  as  long  as  desirable  in  an 
apparatus  illustrated  in  Fig.  82. 

C.    THE  CLARIFICATION  OF  THE  PREPARATION. 

Many  sections  are  quite  too  opaque  for  certain  investigations 
be  they  prepared  ever  so  carefully  and  with  never  so  much 
skill.  They  must  therefore  be  subjected  to  treatment  which 
shall  give  them  the  desired  transparency.  We  call  this  clari- 
fying the  specimen.  The  process  is  usually  applied  to  those 
sections  which  have  many  untransparent  elements,  and  consists 
of  dissolving  out  these  substances  by  means  of  reagents.  Since 
most  of  these  clarifying  substances  are  strong  alkalies  or  acids, 
it  follows  that,  relatively,  but  a  few  classes  of  objects  can  bear 
the  process,  and  of  these  only  those  which  'shall  be  examined 
as  to  the  coarser  histological  relations  of  their  tissue.  We  should 
say  that  this  method  of  clarifying  is  inapplicable  to  all  those 
objects  which  are  to  be  investigated  as  to  some  soft  cell-contents 
or  some  tine  cell- wall  structure. 

There  are  some  substances  which  produce  clarification  very 
gradually.  Glycerine  is  known  to  be  such  a  substance,  and  I 
may  add  also,  according  to  my  experience,  carbolic  acid  and 
creosote.  Sections  which  have  lain  for  a  longtime  in  glycerine 
become  gradually  more  and  more  transparent.  Sections  of 
stigma  tissue  and  the  ends  of  roots  after  lying  in  carbolic  acid 
for  four  weeks  had  become  so  transparent  as  to  be  almost  im- 
perceptible when  put  under  the  microscope. 

But  wTe  usually  adopt  means  which  produce  immediate  clari- 


THE  CLARIFICATION  OF  THE  PREPARATION.  199 

fication.  It  is  a  well-known  fact  that  potassium  hydroxide  in 
a  weak  aqueous  solution  dissolves  protoplasm,  or  at  least  changes 
it  into  a  transparent,  homogeneous  mass.21  It  is  evident  that 
this  can  he  employed  as  a  clarifying  medium  and  all  the  more 
so  since  it  exercises  a  clarifying  influence  upon  the  cell  walls. 

Hanstcin22  was  the  first  to  employ  potassium  hydroxide  to  any 
great  extent  as  a  clarifying  medium,  and  he  did  it  in  investiga- 
tions of  the  merismatic  tissue  of  the  vegetative  point  and  tho 
development  of  the  germ. 

Delicate  sections  need  to  be  immersed  hut  a  few  moments  in 
the  weak  alktili  solution,  washed  out  and  put  in  glycerine  where 
they  become  perfectly  transparent.  The  glycerine,  however, 
should  not  be  used  in  a  concentrated  form  but  diluted  with  al- 
cohol or  water.23  "Thicker  sections  require  longer  treatment  with 
the  potash  solution  and  subsequent  washing  in  hydrochloric  or 
acetic  acid  and  ammonia.  The  section  may  easily  become  too 
transparent  so  that  the  cell  wall  can  no  longer  be  recognized. 
By  using  a  weak  solution  of  alum  they  again  become  distinctly 
visible  and  we  get  in  this  way  the  best  preparations  "  (Han- 
stein)  . 

The  manipulation  in  this  method  of  Hanstein,  which  best 
commends  itself  on  account  of  its  general  suitableness,  is  the 
following.  The  sections  to  be  bleached  are  first  treated  to  a 
weak  solution  of  potassium  hydroxide  in  a  porcelain  vessel  or 
on  a  slide,  for  a  few  seconds,  or  even  some  minutes  according 
to  circumstances,  and  then  carefully  washed  in  distilled  water. 
Hydrochloric  or  acetic  acid  is  used  as  a  neutralize!'  (sometimes 
one  and  sometimes  the  other),  the  section  washed  out  again  and 
then  laid  for  a  short  time  in  weak  liquid  ammonia. 

When  a  tissue  that  is  to  be  clarified  contains  resin  and  fat  mass- 
es along  with  protoplasmic  substances,  a  method  first  suggested 
by  Pfeffer,24  for  making  it  transparent,  may  be  successfully 

21  Sachs,  Lehrb.,  Ill  Auflage,  p.  42. 

22  Hanstein,  Die  Scheitelzellgruppe  in  Vegetationspunct  der  Phanerogamen  (Festschrift 
die  Niederrh.  Ges.  f.  Natur.-ti.  Hielk  z.  Jubilnum  der  Univer.  Bonn,  18G8). — Hanstein,  Die 
Entwicklung  des  Kernies  der  Monokotyleu  uiid  Dikotyleu  (Bot.  Abh.  hernusgegeu  v.  Hans- 
tein, Bd.  I,  Hell  1,  1870). 

23  Hanstein,  Ent\v.  d.  Keimes,  p.  5. 

24 1'tuffer.  Die  Entwicklung  des  Keimes  der  Gnttung  Selaginella  (Botan.  Abh.  heraus- 
gegeu  v.  Hansteiii,  Bd.  I,  Heft  A,  p.  35). 


200  THE  MICROSCOPE  IN  BOTANY. 

employed.  The  preparation  is  laid  for  a  short  time  in  moder- 
ately concentrated  potassium  hydroxide.  Wash  this  out  imper- 
fectly and  repeatedly  add  absolute  alcohol.  A  considerable 
quantity  of  the  fat  will  be  dissolved  in  it,  as  will  also  the  resins 
and  those  substances  which  are  produced  by  the  effect  of  the 
alkali.  The  tissue  which  is  much  collapsed  will  often  swell  out 
again  perfectly  by  the  repeated  addition  of  water,  and  especially 
so  when  the  potassium  hydroxide  was  not  fully  washed  out  with 
the  water,  since  the  potassium  carbonate,  which  always  exists 
in  the  reagent  and  is  insoluble  in  alcohol,  would  be  precipitated 
in  the  cells.  The  preparation  should  now  be  put  in  water  con- 
taining a  very  little  muriatic  acid,  and  we  have  an  object,  which, 
if  the  effect  of  the  alkali  has  been  rightly  regulated,  leaves 
nothing  to  be  desired. 

Recently  Russow25  has  recommended  the  so-called  potassium 
alcohol  as  a  bleaching  medium.  This  is  produced  by  mixing 
absolute  alcohol  with  the  concentrated  solution  of  potassium 
hydroxide,  the  latter  being  added  till  a  little  precipitate  is 
produced.  It  should  be  shaken  frequently  and  left  to  stand 
twenty-four  hours.  The  resulting  pale  yellow,  clear  fluid  should 
be  poured  off  from  the  settlings  and  must  be  diluted  with  dis- 
tilled water  before  using  (2:1).  The  potassic  alcohol  is  ap- 
plied in  the  same  way  as  the  potassium  hydroxide  solution  in 
Hanstein's  bleaching  process.  It  is  to  be  preferred  to  the 
aqueous  alkali  solution  because  the  cell  walls  are  not  so  much 
swollen  in  it  as  in  that. 

[A  serviceable  and  easily  made  bleaching  fluid  is  prepared  by 
the  following  methods :  To  half  a  litre  of  distilled  water  add 
about  50  grammes  of  fresh  chloride  of  lime.  Shake  thoroughly 
and  while  the  lime  is  in  partial  suspension,  add  to  it  a  saturated 
solution  of  common  washing  soda  (carbonate  of  soda)  until  it 
becomes  thick  and  turbid.  Allow  it  to  stand  until  thoroughly 
settled  when  the  clear  supernatant  liquid  should  be  drawn  off 
with  a  siphon  and  kept  in  a  well-stoppered  bottle  in  a  dark 
place.] 

[What  seems  to  be  a  practical,  and,  for  delicate  sections,  a  very 
desirable  method  of  bleaching  is  proposed  by  Mr.  Sylvester 

25  Mern.  de  1'Academie  de  St.  Petersbourg,  Vile  Ser.,  t.  XIX,  No.  1,  p.  15. 


THE  CLARIFICATION  OF  THE  PREPARATION.  201 

Marsh.*  Take  two  wide-mouthed  bottles,  holding  each  about 
75  cc.  Fit  corks  to  them  and  connect  them  by  a  glass  tube 
bent  in  such  a  way  that  it  will  pass  very  nearly  to  the  bottom 
of  one  bottle  but  only  just  below  the  cork  of  the  other.  The 
cork  having  the  long  leg  of  the  tube  should  either  also  be  per- 
forated in  another  place  or  a  notch  cut  in  its  side,  so  as  to  permit 
a  free  passage  of  air  from  within.  Now  fill  one  bottle  nearly 
full  of  distilled  water  into  which  put  the  sections  to  be  bleached. 
Put  in  the  cork  with  the  long  end  of  the  tube.  Cover  the  bot- 
tom of  the  other  bottle  with  chlorate  of  potash  in  crystals  and 
pour  over  it  5  cc.  of  strong  hydrochloric  acid  and  stop  the  bottle 
with  the  cork  having  the  short  end  of  the  bent  tube.  The 
yellow  vapor  of  chlorine  (or  euchlorine)  evolved,  immediately 
passes  over  by  the  tube  into  the  water  containing  the  sections. 
When  the  water  becomes  saturated  the  excess  rises  and  escapes 
through  the  opening  made  in  the  cork.  The  escaping  fumes 
may  be  got  rid  of  by  setting  out  of  doors,  on  the  outside  ledge 
of  the  window  frame  for  instance.  When  the  bleaching  is  car- 
ried far  enough — and  it  needs  to  be  watched — the  washing  may 
be  done  automatically  as  Mr.  Marsh  proposes.  Put  the  sections 
into  a  bottle,  into  the  cork  of  which  a  small  funnel  is  fitted, 
and  which  cork  is  also  provided  with  a  small  orifice  for  the 
escape  of  the  waste  water.  Now  attach  a  small  rubber  tube  to 
the  lower  end  of  the  funnel  which  will  reach  to  the  bottom  of 
the  bottle.  Put  filtering  paper  into  the  funnel  and  set  the  bottle 
where  a  small  stream  of  water  may  run  into  it.  This  is,  of 
course,  filtered  and  passes  quite  down  to  the  bottom  of  the  bot- 
tle and  only  escapes  again  when  it  has  risen  to  the  top.  The 
washing  may  be  done  very  thoroughly  if  one  has  time  to  wait 
f)r  it.  The  advantages  claimed  for  this  method  are  that  the 
sections  are  effectively  bleached  without  being  subjected  to  the 
destructive  and  disintegrating  action  of  the  chlorinated  soda 
solution.  And  the  sections  will  not  suffer  from  a  deposit  upon 
them  of  a  scum  of  carbonate  of  lime,  as  often  happens  when  the 
common  bleaching  fluids  are  used.] 

[E.  Warming  finds  carbolic  acid  a  valuable  reagent  in  reu- 

*  Quoted  from  the  English  Mechanic  by  A.  L.  Wood\vard,  in  American  Monthly  Micro- 
scopical Journal,  Jan.,  1881,  pp.  8, 9. 


202  THE  MICROSCOPE  IN  BOTANY. 

dering  bacteria  transparent.  No  doubt  it  would  be  found  like- 
wise serviceable  with  many  other  kinds  of  vegetable  tissue, 
both  for  clarifying  and  for  mounting.  Its  properties  and  rela- 
tions to  vegetable  histology  have,  apparently,  been  but  little 
studied".] 

[ The  Alcohol  and  Nitric  Acid  Method.*  Place  the  sections 
in  a  watch-glass,  add  alcohol  of  36°  into  which  pour  drop  by 
drop,  concentrated  nitric  acid  until  the  red  vapors  of  the  hypo- 
nitric  acid  are  disengaged.  If  the  preparations  are  violently 
attacked  cover  the  watch-glass  with  a  bell  glass  and  watch  tho 
process.  As  soon  as  the  preparations  rise  to  the  surface  of  the 
mixture,  raise  the  bell  glass  and  by  means  of  two  wooden 
needles  push  them  to  the  bottom  of  the  liquid.  When  there  is 
no  disengagement  of  red  vapor  at  the  normal  temperature,  set 
fire  to  the  alcohol  in  order  to  concentrate  it  further,  and  warm 
the  watch-glass  on  a  piece  of  wire  gauze  over  a  gas  burner. 
Under  these  conditions  the  cell  walls  undergo  a  considerable 
thinning  and  all  their  contents  disappear.  They  become  so 
delicate  that  the  difficulty  is  to  remove  them  from  the  fluid  in 
which  they  have  been  treated  to  the  glycerine  on  the  slide. 
This  may  be  done,  however,  by  adding  to  the  still  warm  alcohol 
a  little  chloroform.  This  hardens  the  sections,  when  by  means 
of  wooden  patula  they  may  be  transferred  to  the  glycerine 
when  they  will  again  soften.  This  process  is  recommended  for 
sections  which  are  to  be  photographed.] 

\_Chromicacid,  according  to  v.  Hohnel,  gives  transparency 
to  the  cell  walls  of  cork,  epidermis  and  cuticular  tissue  and 
the  envelopes  of  pollen  grains.] 

[Calcium  Chloride.  When  it  is  desired  to  give  transparency 
to  the  preparation  without  thinning  it,  especially  if  the  tissue 
is  very  young,  it  is  well  to  use  the  process  followed  by  Treubf 
and  afterwards  by  FlahantJ  and  described  by  the  latter  as  fol- 
lows. Put  the  sections  in  a  watch-glass,  or  in  a  small  porcelain 
capsule  with  one  or  two  drops  of  water.  The  drop  is  covered 

*  Recherches  suv  1'appareil  tegumentaire  des  racines,  Paris,  1881,  L.  Olivier.  Quoted 
in  Jour.  Roy.  Micros.  Soc.,  Vol.  Ill,  No.  5, 1883,  p.  744. 

|  Le  meresteme  primitif  de  la  racine  monocotyledones,  Leyde,  1876. 

J  Recherches  sur  1'accroisement  terminal  de  la  racine  chez  les  Phanerogames,  Ann.  Sc. 
Nat.  VI  (1878),  p.  24.  Quoted  iu  the  Jour.  Roy.  Micros.  Soc.,  Vol.  Ill,  No.  5,  p.  744. 


PREPARATIONS  OF  FOSSIL  PLANTS.  203 

with  a  little  dry  calcium  chloride  in  powder,  and  slowly  warmed 
over  a  small  flame  until  the  desiccation  is  nearly  completed,  and 
then  withdraw  it  from  the  flame  and  add  a  few  drops  of  water  to 
dissolve  the  calcium  chloride.  The  sections  will  now  float  on 
the  water.  They  may  now  be  placed  in  glycerine  in  which  in 
a  few  hours  they  will  be  sufficiently  transparent.  This  treatment 
does  not  dissolve  the  cell  contents  but  darkens  it  by  slightly 
thickening  the  original  very  thin  walls.  The  walls  become  at 
the  same  time  clear  and  brilliant.  The  opacity  of  the  cell  con- 
tents obstructs  the  sight  if  several  layers  are  viewed  at  once. 
A.  B.  H.  | 

VI.     PREPARATION  OF  MICROSCOPIC  SPECIMENS 
OF  FOSSIL  PLANTS.26 

Fossil  plants  occur  in  so  many  different  ways  that  the  methods 
of  their  preparation  for  use  in  microscopical  investigations  must 
be  various.  Sometimes  indeed  they  occur  in  a  condition  such 
that  they  need  no  other  preparation  than  do  the  like  recent 
forms.  They  may  be  used  either  without  further  preparation, 
as  microscopical  objects,  or  they  may  be  got  ready  by  macer- 
ation on  incineration,  or  by  making  thin  sections.  Since  these 
manipulations  have  already  been  described  in  another  place  we 
shall  contine  ourselves  here  to  giving  illustrations  of  them  by 
some  examples  of  their  application  to  the  fossil  flora. 

In  the  first  category  among  others  belong  the  Diatomacece, 
which  in  many  periods  of  the  earth's  development  have  occurred 
in  such  vast  numbers  of  individuals  —  though  of  fewer  spe- 
cies—  that  they  really  enter  into  the  formation  of  the  rocks. 
All  those  fossils  which  are  designated  polishing  powder,  tripoli, 
mountain  meal,  siliceous  marl,  etc.,  consist  in  by  far  the  greater 
part  of  the  siliceous  frustules  of  bacillnria.  These  are  suffici- 
ently prepared  for  microscopical  examination,  when  a  little  of 
the  substance  is  taken  up  on  a  needle  or  lancet  and  placed  upon 
a  slide,  moistened,  and  a  cover-glass  put  over  it. 

A  good  example  of  the  maceration  piocess  is  afforded  by  peat 

26  Since  I  have  had  no  experience  in  preparing  microscopic  specimens  of  fossil  plants, 
my  dear  friend,  Director  Dr.  Hugo  Conwentz  in  Danzig,  has  kindly  undei taken  to  prepare 
this  section. 


204  THE   MICROSCOPE   IN  BOTANY. 

and  stone  coal.  Goppert,  as  long  ago  as  the  year  1836,  em- 
ployed a  method27  by  which  vegetable  remains  could  be  detected 
in  the  densest  varieties  of  coal.  He  treated  the  coal  first  with 
nitric  acid,  in  order  to  prevent  the  potash  salts  melting  together 
with  the  siliceous  earth  in  heating.  He  then  burned  the  coal 
and  treated  the  ashes  with  acids.  The  residuum,  when  ex- 
amined afterwards  with  the  microscope,  was  found  to  contain 
more  or  less  silicated,  epidermal  cells,  tubes  with  simple  and 
bordered  pits,  scaleform  tissue,  etc.,  either  partly  or  wholly 
preserved.  As  Goppert  succeeded  in  detecting  plant  cells  in 
the  compactest  kind  of  coal,  even  anthracite  from  the  graywack 
of  Leibschiitz  in  upper  Silesia,  what  might  we  not  naturally 
expect  to  obtain  by  a  direct  examination  of  the  specimen  itself? 
Very  often  ducts  with  alternating  dots  are  found  among  the 
organic  remains  to  which  Goppert  had  already,  in  1838,  directed 
attention.  They  belong  to  a  coniferous  wood,  Arancarites  car- 
bonarius  G.,  which  occupies  an  important  place  in  the  coal  forma- 
tion and  almost  exclusively  furnishes  the  material  for  the  "fibrous 
coal"  of  the  mineralogist  (mineralogical  wood  coal,  Werner). 
On  the  upper  surface  of  this  layer  of  coal  the  wood  cells  may 
be  recognized  by  their  fine  striated  velvety  appearance,  while 
in  the  interior  they  are  invisible  on  account  of  the  solid  texture 
of  the  coal.  In  recent  times  Count  Fr.  Castracane28  has  found 
diatoms  in  Liverpool  coal  by  the  application  of  a  method  like 
that  of  Goppert.  He  pulverized  it  and  at  a  red  heat  exposed 
it  to  a  current  of  oxygen,  and  macerated  the  decarbonized 
powder  according  to  the  methods  of  Schultze  already  described 
above.29  One  can  apply  this  method  to  many  kinds  of  stone 
coal  and  peat.  A  portion  of  it  should  be  pulverized  and  then 
boiled  with  a  mixture  of  potassium  chlorate  and  nitric  acid, 
and  then  treated  with  water  and  dilute  aqueous  caustic  ammonia, 
and  at  last  with  alcohol  as  long  as  any  soluble  material  can  be 
extracted  from  it.30  When  the  residuum  is  examined  with  the 
microscope,  isolated  organic  parts  may  be  recognized,  as  fern 
spores,  the  remains  of  sporangia,  etc. 

27  Goppert,  Die  fossilen  Farnkrauter,  Breslau  und  Bonn,  1836,  p.  XVIII. 

28  Priugsheim's  Jahrb.,  iiir  Wissensch.,  Botanik,  Bd.  X,  p.  l,jf. 

29  See  page  163. 

30  Verhaudl.,  der  Berliner  Akademie  der  Wissenschaften,  1835. 


PREPARATIONS  OF  FOSSIL  PLANTS.  205 

Many  of  the  bituminous  woods  embedded  in  the  alluvium, 
diluvium,  and  in  the  tertiary  strata  are  so  well  preserved  that 
transverse  and  longitudinal  sections  may  be  made  from  them  in 
the  way  usually  adopted  for  recent  woods.  With  others,  on  the 
contrary,  it  is  recommended  to  moisten  the  .cutting  surface,  not 
with  water,  but  with  a  dilute  solution  of  potash,  to  prevent  the 
section  from  falling  apart.  In  the  same  way  we  frequently 
obtain  suitable  sections  of  the  large  pieces  of  amber  embedded  in 
the  wood.  But  in  case  the  solidity  of  the  wood  has  been  at  first 
disturbed,  and  the  structure  of  it  loosened,  it  should  be  em- 
bedded for  half  a  day  or  longer  in  a  thick  solution  of  gum  and 
then  the  desired  section  can  be  made.  In  most  cases  it  will  be 
well  to  treat  the  section  to  alcohol  to  clear  it  up. 

In  contradistinction  to  the  above  mentioned  fossils,  the  great 
majority  of  such  plants  require  a  treatment  differing  entirely 
from  this,  namely,  grinding  smooth  and  grinding  thin.  To  this 
group  belong  on  the  one  side  the  series  of  fossil  resins,  especi- 
ally amber,  and  on  the  other,  woods  which  by  various  means 
have  become  petrified.  When,  for  example,  one  obtains  a  piece 
of  amber  containing  blossoms  or  parts  of  blossoms  which  he  can 
examine  with  a  low  power  he  will  only  need  to  grind  it  to  a  flat 
surface.  But  if  there  is  inclosed  some  form  of  fungus,  pollen, 
or  wood  tissue  which  must  be  more  strongly  magnified  the 
preparation  of  a  thin  section  becomes  indispensable.  Petri- 
fied woods  should  be  ground  thin  only  when  they  can  then  be 
examined  by  transmitted  light,  that  is  to  say,  when  they  are 
petrified  by  such  substances  as  in  very  thin  sections  possess  a 
certain  pellucidity.  For  example,  woods  which  have  been  meta- 
morphosed in  marcasite,  copper  ore,  etc.,  cannot  be  examined 
with  transmitted  light,  but  must  be  illuminated  from  above. 

Most  of  the  petrified  woods  —  and  there  are  in  the  different 
strata  from  the  Devonian  to  the  Oligocene,  a  very  great  num- 
ber of  them — require  a  special  kind  of  preparation  to  make  it 
possible  to  examine  them  with  transmitted  light.  With  many, 
particularly  coniferous  woods,  it  may  be  done  by  a  skilfully 
struck  blow,  preferably  in  a  .radial  direction,  splitting  off  thiii 
splinters,  which,  without  further  preparation,  may  be  applied  to 
the  desired  purpose.  In  order  to  increase  this  transparency 


206  THE  MICROSCOPE  IN  BOTANY. 

they  may  be  inclosed  in  water  or  Canada  balsam  and  placed 
under  a  cover-glass. 

It  is  very  difficult  to  split  off  a  sufficiently  thin  splinter  in  a 
tangential  direction,  and  in  a  horizontal  it  is  quite  impossible. 
Since  a  knowledge  ,of  the  three  named  aspects  of  the  wood  is 
necessary  in  order  to  determine  to  what  particular  order  it  be- 
longs, thin  sections  cannot  very  well  be  dispensed  wilh.  But 
in  many  cases,  when  the  inquiry  only  seeks  to  determine  if  the 
wood  be  coniferous  or  deciduous,  the  test  can  be  adequately 
made  with  the  thin  splinter.  The  preparation  of  ground  sections 
in  general  was  first  made  with  fossil  wood  by  W.  Nicol,31  and 
was  then  published  by  Witham  in  the  "  Observations  of  fossil 
vegetables,  London,  1831."  Independently  of  this  Goppert 
and  linger  in  Germany  invented  separately  a  like  method,  by 
which  they  ground  small  splinters  very  thin,  while  hitherto 
petrified  wood  had  been  ground  upon  one  side  only,  and  had 
been  examined  with  reflected  light.  This  method  was  greatly 
modified  by  the  authors  themselves,  as  it  has  been  by  others  till 
now  scarcely  two  naturalists  use  exactly  the  same  method 
throughout  in  the  preparation  of  ground  sections. 

It  should  be  incidentally  remarked  that  the  attempt  was  after- 
wards made  to  prepare  ground  sections  of  rocks  in  order  to 
examine  their  structural  relations  with  the  microscope.  This 
method  was  founded  in  1858,  by  Sorby's  treatise  "On  the  micro- 
scopical structure  of  crystals  indicating  the  origin  of  minerals 
and  rocks,"32  and  in  Germany  Zirkel  deserves  the  credit  of 
having  done  good  services  in  this  direction  by  his  "Microscop- 
ical studies  of  stone."33  Through  him  and  others,  the  micro- 
scopical investigation  of  rocks  has  been  so  far  advanced  that  it 
has  led  to  a  complete  transformation  of  petrography.  The 
microscope  has  become  to-day  an  indispensable  instrument,  not 
only  for  the  botanist  and  zoologist,  but  for  the  geologist  also. 

The  way  and  manner  of  preparing  ground  sections  can  be 

81  Further  information  concerning  this  subject  can  be  found  in  the  works  named  below. 
Zirkel,  Die  Mikros.  Besehaffenheit  der  Mineral ien  und  Gesteine,  Leipsig,  1873.—  Rosen* 
busch,  Mikroskopische  Physiographic  der  petrographische,  wichtigen  Minernlieu,  Stuttgart, 
1873.— v.  Lasaulx,  Elemente  der  Peirographie,  Bonn,  1875. 

32  Quarterly  Journal  of  the  Geological  Society,  London,  Nov.,  185S,  Vol.  XIV,  p.  453,  ,/F. 

»s  Sitzb.  d.  k.  k.  Acad.  in  Wien,  1803,  Bd.  47,  Abth.  1,  p.  21G,ff. 


PREPARATIONS  OF  FOSSIL  PLANTS.  207 

varied  in  many  ways,  and  we  limit  ourselves  therefore  to  pre- 
senting the  more  general  outlines  of  the  subject. 

1.  Cutting  out  the  Specimen.  In  order  to  be  able  to  pre- 
pare a  thin  section  it  is  first  required  to  cut  off  the  thinnest 
possible  slice  from  the  piece  in  hand.  While  it  might  be  pos- 
sible to  knock  off  such  a  piece  from  a  stone  specimen,  with  a 
skilful  blow  of  the  hammer,  it  would  be  scarcely  possible  to  do 
so  with  petrified  wood,  especially  in  a  direction  transverse  to 
the  original  grain  of  the  wood.  On  this  account  we  adopt  an- 
other method  for  obtaining  the  three  different  sections  of  fossil 
wood.  We  cut  them  off  from  the  specimen.  If  the  material 
has  but  little  consistency  it  were  better  to  saturate  it  previously 
with  Canada  balsam  [first  hardened  and  then  softened  by  warm- 


FlG.  95. 

ing]  in  order  the  more  easily  and  safely  to  take  away  sections  of 
it  which  will  hang  together.  Fig.  1)5  represents  a  small  hand 
cutting-machine.*  The  specimen  or  a  fragment  of  it  is  fastened 
to  the  iron  holder  a,  by  means  of  the  common  stone-grinder's 
cement.  The  holder  rotates  in  two  directions  and  is  movable 
also  on  the  axis  b.  A  weight  c  fixed  to  the  axis  b  presses  the 
specimen  against  the  steel  disk  d  which  is  put  in  rotation  by 
means  of  a  small  wheel  e  having  a  cogged  rim.  With  this  con- 
trivance it  is  clear  that  one  may  cut  thin  pieces  from  the  spec- 
imen, in  different  directions,  without  removing  it  from  its 
fastening.  The  weight  should  not  be  too  heavy,  for  if  the  fossil 

*  This,  and  the  combined  cutting  and  grinding  machine,  illustrated  in  Fig.  97,  are  made 
by  Messrs.  Voigt  and  Hochjjesang  of  Gottingen,  the  former  lor  06  M.=  $  16,  and  the  latter  for 
200  M.=$.">0.  I  am  not  informed  \vliat  firm,  if  any,  in  this  country,  make  or  keep,  for  sale  » 
these  machines.  Doubtless  any  of  the  principal  optical  houses  would  import  them  to  01* 
der.  A.  B.  H. 


208  THE  MICROSCOPE  IN  BOTANY. 

presses  too  hard  against  the  cutting  disk  it  may  be  caused  to 
oscillate  and  break  the  thin  slice  of  stone.  Emery  mixed  with 
water  should  be  used  as  a  cutting  medium.  While  turning  the 
wheel  with  the  right  hand  the  other  maybe  employed  in  putting 
the  emery  on  the  disk  by  means  of  a  hair  pencil.  There  is  also 
arranged  around  the  lower  part  of  the  disk,  a  box  of  sheet  metal 
to  catch  the  ground  material  and  to  prevent  it  also  from  being 
scattered  about  laterally.  This  was  left  off  the  drawing  on  ac- 
count of  its  covering  up  essential  parts  of  the  apparatus.  If 
one  wishes  he  can  so  modify  the  construction  of  this  machine 
as  to  propel  the  large  wheel  by  foot-power  like  a  sewing  machine. 

As  to  the  .size  of  the  thin  piece  it  is  recommended  to  take  a 
surface  about  15  mm.  square  at  the  outset.  It  will  be  somewhat 
reduced  in  size  afterwards,  chiefly  in  consequence  of  grinding 
away  the  edges,  so  that  when  finished  it  will  not,  perhaps,  be 
more  than  12  mm.  square,  which  generally  is  enough. 

The  next  step  is 

2.  Grinding  down -the  Specimen.  By  this  expression  we 
understand  grinding  the  surface  smooth.  If  the  preparation  is 
large  enough  and  of  the  right  consistency  it  may  be  held  in  the 
hand,  otherwise  it  should  be  cemented  to  a  small  glass  plate 
which  serves  as  a  convenient  handle.  Canada  balsam  is  a  suit- 
able cement.  It  should  be  sufficiently  warmed  to  melt  it,  and 
the  specimen  should  also  be  warmed  over  a  spirit  lamp  or  gas 
burner  sufficiently  to  remove  the  moisture,  then  lay  it  on  the 
balsam  and  again  heat  the  plate  till  the  balsam  boils,  being  careful 
not  to  burn  it.  After  the  Canada  balsam  has  cooled  and  the 
development  of  bubbles  has  ceased,  press  the  specimen  down  fast. 
If  bubbles  yet  appear  under  it,  it  will  be  necessary  to  repeat  the 
process,  else  one  may  run  the  risk  of  getting  the  specimen 
broken. 

An  emery  plate  serves  an  excellent  purpose  for  grinding  down 
the  preparation.  It  is  first  ground  on  a  coarse  grained  plate  and 
then  the  surface  is  polished  on  a  finer  one.  Since  the  grinding 
stone  must  be  kept  moist,  it  may  be  laid  in  a  suitable  dish  with 
a  flat  bottom  and  water  enough  poured  in  to  cover  the  upper 
surface  of  the  stone,  Fig.  96.  Should  it  not  be  possible  to 
make  the  specimen  sufficiently  smooth  on  the  fine  grained  stone, 


PREPARATIONS  OF  FOSSIL  PLANTS.  209 

it  may  be  done  on  a  ground-glass  disk  with  emery  which  con- 
tains no  coarse  grains,  or,  better  still,  on  a  whetstone  with  the 
use  of  oil  of  turpentine. 

These  emery  stones  have  but  recently  come  into  the  market. 
x  Formerly  the  grinding  was  done  on  a  cast-iron  plate  by  means 
of  emery  and  water.  The  new  method  has  the  advantage  that 
it  is  simpler,  leads  more  directly  to  the  desired  end,  and  makes 
greater  cleanliness  possible.  According  to  the  experience  of 
others  as  well  as  my  own,  it  may  be  commended  as  the  best. 

3.      Grinding  the  Preparation  thin.     When  one  surface  of 
the  specimen  has  been  ground  and  polished,  it  should  be  cleaned 


FIG.  9U. 


with  a  hair  pencil  and  plenty  of  water  poured  over  it.  Then  it 
should  be  left  in  the  air  to  dry.  Do  not  under  any  circumstances 
undertake  to  hasten  the  process  by  rubbing  with  woollen  or  linen' 
cloth,  for  this  procedure  unavoidably  leaves  small  fibres  upon 
the  preparation  which  very  much  damage  the  microscopic  image. 
It  should  then  be  separated  from  the  glass  plate  by  warming 
the  Canada  balsam  and  again  cemented  down,  this  time  with  the 
smooth  side  upon  the  plate, —  first,  however,  having  dried  out 
any  adhering  moisture  over  a  spirit  lamp.  Now  begins  the  real 
thin-grinding,  first  again  on  a  coarse  grained  emery  plate,  in 
doing  which  precautions  are  to  be  observed.  The  second  sur- 
face is  to  be  ground  uniform  and  parallel  with  the  first,  but  being 
careful  not  to  grind  away  the  edges.  At  last  on  the  fine  grained 
14 


210 


THE   MICROSCOPE  IN   BOTANY. 


stone  when  the  preparation  has  become  very  thin,  special  care 
is  to  be  taken  not  to  grind  the  section  through,  or  break  it,  al- 
though this  docs  sometimes  happen,  even  with  the  most  skilful. 
In  order  to  prevent  the  possibility  of  its  occurrence,  Zirkel 
recommended  that  four  pieces  of  cover-glass  be  fastened  to  the 
four  under  corners  of  the  glass  plate,  and  of  course  the  section 
cannot  easily  become  thinner  than  those. 


FIG.  97. 


In  respect  to  the  thinness  which  the  section  should  be  made 
to  attain,  nothing  in  general  can  be  determined,  it  depending 
rather  in  each  individual  case  on  the  pellucidity  of  the  material. 
If  the  material  has  a  high  degree  of  transparency,  the  prepara- 
tion may  be  ground  only  relatively  thin,  in  order  to  recognize 
clearly  the  structural  relations.  In  other  cases,  when  the  material 
is  almost  entirely  opaque,  it  is  naturally  recommended  to  grind 


PREPARATIONS  OF  FOSSIL  PLANTS.  211 

the  section  as  thin  as  possible.  In  general  one  may  consider  a 
section  sufficiently  thin  when  one  may  see  through  it  to  read 
after  having  moistened  it  with  water. 

In  order  to  save  time  and  trouble  one  may  fasten  several 
specimens  on  the  glass  plate  and  grind  them  all  at  once.  To 
do  that,  indeed,  will  require  that  they  should  be  ground  to  a  like 
thickness,  and  about  equally  withstand  the  action  of  the  emery 
stone. 

The  process  of  preparing  the  section  may  be  lightened,  and 
time  and  care  saved  by  the  use  of  a  machine  which  will  rotate 
the  disk  while  the  specimen  is  held  stationary  instead  of  the 
other  course  as  has  now  been  shown.  This  is  recommended  by 
Rosenbusch  and  others.  But,  on  the  contrary,  Zirkel  in  his 
extensive  works  does  not  approve  of  it.  A  larger  machine 
has  been  constructed  by  the  above  mentioned  firm  to  be  driven 
by  foot  power  and  which  can  be  used  for  both  cutting  and  grind- 
ing the  section.  Fig.  97  gives  an  illustration  of  this  com- 
bined machine.  The  cutting  arrangement  is  essentially  the  same 
as  described  above ;  a  is  the  carrier,  b  the  movable  axis  for  this 
and  the  weight  c,  d  is  the  cutting  disk,  e  a  small  driving  wheel 
which  is  connected  with  the  balance  wheel  f,  by  an  endless  band. 
The  treadle  g  moves  this  wheel,  k  represents  the  folding  guard 
box.  The  cast  iron  disk  m  by  which  the  grinding  is  done  runs 
on  the  vertical  spindle  7,  in  a  depression  in  the  table,  by  means 
of  a  belt  from  a  second  wheel  i  running  over  the  guide  pulley  k. 
Probably  this  contrivance  might  be  so  modified  as  to  allow  an 
emery  plate  to  be  substituted  for  the  iron  one. 

4.  Mounting  the  Preparations.  Since  the  glass  plate  is 
commonly  larger  and  stronger  than  the  object-slide  used  for  the 
preservation  of  microscopic  sections,  and  since  also  it  is  much 
ground  and  scratched  by  the  process,  the  removal  of  the  speci- 
men when  the  grinding  is  finished  is  indispensable.  This  is 
often  a  matter  of  great  difficulty  and  must  be  done  by  observ- 
ing those  precautions  which  are  particularly  set  forth  on  page 
197  of  this  work.  When  the  specimen  has  been  successfully 
embedded  in  Canada  balsam,  put  on  a  cover-glass  and  warm  the 
slide  again  carefully  and  press  down  the  cover.  After  the  spe- 
cimen has  become  perfectly  cold,  the  superfluous  balsam  should 


212 


THE  MICROSCOPE  IN  BOTANY. 


be  removed  with  a  knife  from  about  the  edges  of  the  cover  and 
the  slide  perfectly  cleaned  with  alcohol  and  water. 

It  still  remains  to  consider  the  labelling  of  the  specimen,  which 
may  be  suitably  done  by  strips  of  paper  pasted  on  above  and 
below  as  shown  in  the  illustration,  Fig.  98.  On  the  upper  one 
write  the  name  of  .the  fossil  and  designate  in  one  corner  the 
catalogue  or  other  number,  and  in  the  other 
the  direction  in  which  the  section  is  made, 
using  the  abbreviations  H.  for  horizontal, 
R.  for  radial,  and  T.  for  tangential.  On 
the  lower  label  should  be  the  rock  formation 
to  which  it  belongs,  the  locality,  the  date 
of  finding,  and  the  name  of  the  collector. 
Besides  this  it  is  advised  to  write  with  a 
diamond,  on  the  back  of  the  slide  opposite 
the  label,  some  inscription  by  which  the 
section  can  be  identified  with  the  specimen 
from  which  it  is  cut,  if  possible ;  for  in 
case  of  the  loss  of  the  label  the  engraved 
note  then  becomes  of  the  utmost  im- 
portance. For  purposes  of  identification,  indeed,  it  is  sufficient 
to  engrave  the  catalogue  number. 

5.  Preservation  of  the  Preparations.  Finally,  the  preserva- 
tion of  the  specimens  should  be  referred  to,  since  it  differs  from 
the  method  applied  to  other  microscopic  sections.  The  latter, 
so  far  at  least  as  they  are  mounted  in  a  fluid,  must  be  kept  in  a 
horizontal  position,  and,  if  desirable,  provided  with  a  protecting 
ledge.  This  is  not  required  with  ground-sections  and  so  from 
this  fact  one  may  determine  what  suitable  arrangements  should 
be  made.  Commonly  it  is  done  in  this  way.  The  specimens 
are  stored  in  wooden  boxes  provided  with  rack  supports,  whose 
distance  apart  corresponds  to  the  size  of  the  slide  used. 

I  have  recently  contrived,  for  the  preservation  of  microscop- 
ical ground-sections,  for  the  West  Prussia  Provincial  Museum 
in  Danzig,  a  tray  which  rests  upon  a  somewhat  different  prin- 
ciple. Its  essential  point  is  this,  that  the  slide  rests  upon  its 
short  edge  in  a  sloping  position,  thus  .making  it  much  more  con- 
venient to  read  the  label.  Besides,  one  can  thus  store  in  one 


FIG. 


THE  PREPARATION  OF  PERMANENT  MOUNTS.  213 

tray,  and  have  under  his  eye  at  once,  a  very  great  number  of 
specimens.  It  is  42  X  46  cm.  in  size,  and  exactly  fits  into  the 
drawers  of  those  cases  which  are  intended  for  microscopical, 
palasontological  and  geological  collections.  Two  to  four  of  these 
trays,  according  to  the  depth  of  the  drawer,  may  be  placed 
over  each  other.  Thus  from  eight  to  sixteen  hundred  of  these 
ground-sections  may  be  opened  for  inspection  in  one  drawer. 
The  contrivance  is  illustrated  in  Fig.  99.  A  simplification  of 
this  plan  might  perhaps  be  made  by  substituting  for  each  of  the 
forty  sloping  transverse,  cross  ledges,  in  the  two  rows,  two 
longitudinal  ledges  with  sloping  grooves  cut  in  them. 


FIG.  99. 


VII.   THE  PREPARATION  OF  PERMANENT  MOUNTS. 

Having  already  learned  how  an  object  should  be  prepared  to 
be  examined  by  the  microscope,  and  having  also  particularly 
illustrated  the  different  methods  of  preparing  an  object,  we 
will  here  describe  the  preparation  of  permanent  mounts.  Per- 
manent preparations  are  such  as  are  preserved  for  a  long  time 
in  a  condition  to  be  taken  at  any  moment  and  subjected  to  ex- 
amination. 


214  THE  MICROSCOPE   IN   BOTANY. 


1.    OBJECT-SLIDE  AND  COVEK-GLASS. 

It  has  already  been  several  times  mentioned  that  the  prepa- 
ration to  be  examined  must  lie  upon  a  glass  plate  in  fluid,  and 
commonly  be  covered  with  a  piece  of  thin  glass.  In  the  per- 
manent preparation  both  glasses  are  always  required.  The 
under,  stronger  glass  bears  the  name  of  the  object-carrier 
(object-slide),  the  other,  the  thin  plate,  the  cover-glass. 

A,  TIte  Object-slide,  is  a  plate  made  from  pure  mim>r  glass, 
often  also,  of  common  green  window-glass.  The  most  suit- 
able thickness  is  from  1  to  1.5  mm.  Glass  thinner  than  1 
mm.  is  so  thin  as  to  be  easily  broken,  and  such  as  is  over  2  mm. 
is  too  thick  for  this  use.  Perfectly  colorless  glass  is  much  to 
be  preferred,  though  glass  with  a  slight  green  tinge  may  be 
used  without  harm.  It  is  much  more  important  that  the  slide, 
especially  where  the  object  lies,  should  be  free  of  all  minute  air 
bubbles,  flaws  and  scratches. 

The  edges  should  not  be  rough.  They  can  be  ground  by  the 
glazier  without  difficulty,  or  one  can  do  it  for  himself  on  the 
whetstone  with  turpentine  oil. 

What  form  to  give  the  slide  is  purely  a  matter  of  taste,  only 
they  should  be  neither  too  large  nor  too"  small.  Those  sizes  which 
have  come  into  most  general  use  are  the  following : 

(a)  The  English  form,  76  mm.  long  X  26  mm.  broad,  Fig. 
100,  I. 

(b)  The  German  or  Giessener  society  form,  48  X  28  mm., 
Fig.  100,  I,  II. 

(c)  The  Vienna  form,  65  X  25  mm.,  or  70  X  27  mm,  Fig. 
100,  II." 

The  form  most  used  in  Germany  is  form  b.  We  prefer,  how- 
ever, form  a  to  all  others.  [This  is  the  one  by  far  the  most 
generally  used  in  this  country,  only  one  firm  offering  for  sale 
another  size,  60  X  19  mm.  A.  B.  H.] 

Before  using  the  slide  it  is  necessary  to  clean  it  very  carefully. 
This,  in  most  cases,  may  be  satisfactorily  done  by  washing  in 

s*  Still  other  and  less  useful  forms  75X30  mm.,  70X35  mm.,  70X30mm.,  60X^5  mm., 
33X33  mm.,  the  latter  for  stone  sections,  are  also  sold  by  opticians  In  Germany. 


THE  COVER-GLASS. 


215 


water  and  alcohol  and  rubbing  dry  with  a  clean  cloth.  For 
very  dirty  slides,  or  when  one  desires  to  have  an  absolutely 
clean  surface,  the  following,  although  somewhat  detailed,  pro- 
cess is  recommended.  Lay  the  slide  in  a  porcelain  saucer  with 
hot  nitric  acid.  Take  it  out  after  a  few  minutes  with  the  for- 
ceps and  pour  distilled  water  over  it.  Then  for  a  short  time 


HL 


FIG.  100. 

put  it  in  a  weak  potash  solution.  Then  wash  with  distilled 
water  till  the  alkali  is  entirely  removed.  Pour  over  it  absolute 
alcohol  and  finally  cover  it  with  ether  and  let  this  evaporate. 
By  this  process  we  get  a  perfectly  clean  surface,  as  bright  as  a 
mirror,  without  the  use  of  a  cloth. 

B.  The  Cover-glass.  The  thickness  of  the  cover-glass,  as  is 
well  known,  has  its  influence  upon  the  microscopic  image.  Fol- 
low and  medium  powers  the  cover- glass  may  be  from  0.2  to 


216  THE  MICROSCOPE  IN  BOTANY. 

0.4  mm.  thick,  for  still  higher  and  the  highest  powers,  the  thick- 
ness should  never  be  more  than  0.15  to  0.10  mm.,  or  still  less 
than  that. 

The  form  of  the  cover-glass  is  in  our  judgment  of  consider- 
able moment  to  the  usefulness  and  durability  of  the  preparation. 
There  are  circular,  square,  and  rectangular  oblong  cover-glasses 
of  various  sizes.35 

One  should  make  it  a  rule  never  to  use  too  small  a  cover- 
glass,  either  in  the  permanent  preparations,  or  in  those  which 
are  preserved  only  for  present  temporary  use.  Otherwise,  in 
fresh  preparations,  one  runs  the  risk  of  having  the  fluid  em- 
ployed exude  around  the  cover-glass  and  come  in  contact  with 
the  objective,  and  in  permanent  mounts  we  find  the  cement  ring 
around  the  edge  of  the  glass  seriously  interfering  with  the  focus- 
sing in  the  use  of  high  powers.  For  common  use  squares  of 
18  mm.  on  the  side,  and  circles  of  15  mm.  in  diameter  are  per- 
haps best.  We  give  the  circles  the  preference  before  all  others 
in  mounting  permanent  preparations,  since  they  are  by  far  th ) 
easiest  to  cement  perfectly  tight.  One  naturally  should  employ 
the  largest  possible  cover-glass  in  those  investigations  where 
reagents  are  used,  which,  it'  they  come  in  contact  with  the  ob- 
jective are  liable  to  injure  it,  or  which  may  develop  an  injurious 
vapor  upon  it. 

[For  cleaning  slides  and  cover-glasses,  and  for  a  convenient 
way  to  keep  the  latter  so  that  they  will  be  most  easily  accessible 
for  immediate  use,  I  know  of  nothing  better  than  the  plan  sug- 
gested by  Mr.  C.  E.  Hanaman  of  Troy,  N.  Y.,  some  years  ago.* 
It  is  as  follows  :] 

[A  solution  long  in  use  by  photographers  for  cleaning  their 
negative  plates  and  glass  vessels  is  utilized,  as  being  quite  as 
efficacious  as  the  nitric  acid  bath  and  wholly  free  from  its  dis- 
agreeable odors.  The  mixture  consists  of  a  cold,  aqueous  sat- 
urated solution  of  bichromate  of  potash  to  which  is  added  about 
one-eighth  of  its  bulk  of  strong  sulphuric  acid.  The  mixing 
should  be  made  in  a  porcelain  dish,  or  a  thin  glass  vessel,  as  the 

35  In  the  price  current  of  Stender  the  square  ones  are  quoted  of  10, 12,  15,  and  18  mm. 
square,  the  round  ones  ol  6,  8, 10, 12. 15, 18,  22  mm.  iu  diameter,  and  the  oblong,  20X21  >  —X 
16,  18X'2  mm. 

*  American  Naturalist,  Aug.,  1878,  p.  573. 


PRESERVING  MEDIA.  217 

heat  generated  might  break  a  glass  bottle.  The  vessel  should  be 
set  outside  for  the  liquid  to  cool,  after  which  no  more  injurious 
vapors  will  be  given  off.  Then  the  liquid  may  be  kept  for 
future  use  in  a  glass-stoppered  bottle.  The  slides  should  be 
plunged  one  by  one  into  a  porcelain  dish  containing  a  quantity 
of  the  liquid,  till  all  are  put  in,  then  tilt  the  dish  in  such  a  way 
as  to  cause  the  liquid  to  flow  back  and  forth  through  the  mass 
for  a  few  moments,  and  then  after  pouring  off  the  liquid  place 
the  dish  under  a  stream  of  water  from  an  open  tap.  They  are 
then  wiped  dry  with  soft  linen  cloths.] 

[The  cover-glasses,  after  being  treated  with  the  cleaning  liquid, 
are  thoroughly  washed  with  distilled  or  filtered  water,  and  then 
taken  out  with  the  forceps  one  by  one,  and  dried  by  laying  each 
on  one  corner  of  a  soft  linen  cloth  on  the  table  and  gently 
rubbing,  first  one  and  then  the  other  side  with  another  part  of 
the  cloth.  The  cloths  used  for  this  purpose  —  preferably  old, 
worn  and  soft  ones  —  should  be  first  boiled  in  carbonate  of  soda 
and  rinsed  in  hot  filtered  or  distilled  water.  In  order  to  keep 
the  covers  clean,  and  make  them  most  easily  accessible  for  im- 
mediate use,  as  well  as  to  greatly  facilitate  the  selection  of  one 
of  any  desired  thickness,  the  covers  are  finally  arranged  edge 
upwards,  in  a  box  or  drawer,  between  strips  of  thick,  white 
blotting-paper.  The  strips  of  blotting  paper  should  be  cut  two- 
thirds  as  wide  as  the  cover,  should  reach  from  side  to  side  of 
the  drawer  or  box,  and  should  be  separated  at  the  ends  by 
squares  of  the  same  paper,  thus  forming  a  rack  in  which  the 
covers  stand  edge  upward,  and  from  which  they  can  be  readily 
picked  out  (Hanaman).  A.  B.  H.] 

2.    PRESERVING  MEDIA    (Mounting  Fluids). 

The  freshly  prepared  botanical  specimen,  which  is  to  be  sub- 
jected to  microscopical  examination,  is  usually  put  in  a  drop  of 
distilled  water  on  the  slide  and  a  cover-glass  put  over  it.  But 
we  have  already  seen  that  water  is  not  the  only  fluid  to  be 
used  in  examining  freshly  prepared  objects.  So  likewise  in 
permanent  mounts  water  is  scarcely  suitable  to  use,  since  most 
objects  would  in  time  decay  in  it.  We  must  choose,  therefore, 


218  THE  MICROSCOPE  IN  BOTANY. 

those  preserving  media  which  will  keep  the  object  from  decay  j 
and  yet  at  the  same  time  be  sufficiently  indifferent  to  it  as  not 
to  change  the  object  itself  in  the  slightest  degree.  A  large 
number  of  such  media  have  been  brought  into  use,  but  we  shall 
undertake  to  enumerate  only  those  deemed  most  important  and 
most  useful.  They  are  either  evaporating  or  non-evaporating 
fluids  or  substances,  and  those  which  may  be  applied  in  a  fluid 
state  but  which  afterwards  stiffen. 

A.  Glycerine.  For  the  botanist  this  is  the  most  important  of 
all  the  preserving  fluids.  It  may  be  used  with  most  vegetable 
preparations,  but  it  preserves  starch  and  chlorophyll  grains  rela- 
tively the  best.  In  mounting  the  red  algre,  bacteria  and  diatoms, 
it  should  not  be  used  ( Poulsen) .  Concentrated  glycerine  causes 
much  shrinking  of  the  tissue  by  withdrawing  the  water  from  it 
and  a  contraction  of  the  primordial  utricle.  This,  however,  may 
be  prevented  in  great  part  by  first  placing  the  preparation  for  a 
considerable  time  in  a  weak  solution  of  glycerine  and  distilled 
water.  Glycerine  should  be  employed  in  mounting,  either  con- 
centrated or  diluted  with  water,  in  various  proportions  (1:1, 
etc.).  Strong  glycerine  is  often  too  powerfully  clarifying  to 
be  suitable  for  certain  kinds  of  tissue.  It  should  then  be  di- 
luted. Frey  recommended  the  use  of  concentrated  glycerine 
with  the  addition  of  a  small  quantity  of  pure  carbolic  acid.  For 
other  purposes  he  adds  to  30  g.  of  glycerine,  2  drops  of  strong 
hydrochloric  acid  or,  in  place  of  this,  concentrated  acetic  acid. 
Certain  stain  ings  require  the  addition  of  acid,  otherwise  the  color 
will  fade  after  a  time.  According  to  Dippel,  the  addition  of  a 
little  acetic  acid  to  concentrated  glycerine  modifies  the  effect  of 
its  shrinking  properties,  and  its  too  powerful  clarifying  of  thin 
tissue. 

For  the  preservation  of  very  delicate  objects,  green  algae, 
etc.,  a  glycerine  mixture  invented  by  Hantsch^6  is  recom- 
mended, which  Dippel  describes  as  follows  :  "  Three  parts  pure 
90  per  cent  alcohol  with  2  parts  water  and  1  of  glycerine.  In 
order  to  moderate  the  effect  of  this  fluid  as  much  as  possible  at 
the  beginning,  the  object  is  put  in  a  drop  of  water  on  the  slide 
to  which  a  small  drop  of  the  mixture  has  been  added.  Then 

se  Hantsch  in  Reinicke's  Beitrngen  zur  neueren  Mikroskopie,  Heft  III,  p.  37,  /. 


PRESERVING  MEDIA.  219 

the  preparation  is  put  in  some  place  free  from  dust  and  allowed 
to  stand  till  the  water  and  alcohol  have  entirely  evaporated. 
Then  add  another  drop  and  let  it  evaporate  as  before,  and  con- 
tinue this  process  till  the  object  has  as  much  glycerine  as  it 
should  have  to  be  properly  mounted.  In  this  way  the  good 
preservation  of  the  object  is  perfectly  assured.  It  is  advised, 
however,  before  the  mounting  is  finished,  to  let  the  object 
lie  for  some  days  in  order  to  be  fully  convinced  that  the 
preserving  medium  has  no  more  fluid  in  it  that  can  be  evapo- 
rated." 

Objects  to  be  mounted  in  glycerine  should  be  first  moistened 
in  distilled  water  before  they  are  put  into  the  glycerine.  Lay 
the  object  in  a  dish  of  water,  put  a  drop  of  the  glycerine  on  the 
slide  and  then  transfer  the  object  to 
it  with  the  least  possible  amount  ot* 
water  adhering  to  it.  Then  put  on 
the  cover-glass.  It  will  often  be  a 
matter  of  some  difficulty  with  the  be- 
ginner to  know  exactty  how  much  of 
the  glycerine  to  use  in  order  to  fill 
out  the  space  under  the  cover-glass 
and  yet  not  so  much  as  to  cause  it  to 
run  out  around  the  edges.  The  right 
quantity  may  very  nearly  be  determined 
in  the  following  way.  A  bottle  with  a 

Ion o-  stopple,  of  the  form  of  Fig.  101, 

,,       „,,    i       vi       i         •         +1  FlG- 

is   partly    filled   with   glycerine,    the 

stopper  put  in  and  lifted  out :  there  will  always  be  a  drop  of 
the  same  size  hanging  to  its  point.  This  is  put  on  the  slide 
as  an  experiment  and  covered  with  a  glass  of  the  size  commonly 
used.  Now,  if  there  is  too  much  or  too  little  fluid  on  the  slide, 
there  should  be  some  fluid  taken  out  of  or  added  to  the  glyce- 
rine in  the  bottle,  as  the  case  may  be,  till  it  stands  at  exactly 
the  right  place  to  give  a  drop  of  the  required  size.  It  should 
then  be  kept  at  the  same  height. 

Glycerine  should  never  be  shaken  because  it  thus  collects 
small  air  bubbles  which  are  transferred  to  the  preparation.  In 
order  to  free  commercial  glycerine  from  air  bubbles,  warm  it  a 


220  THE  MICROSCOPE  IN  BOTANY. 

little  and  then  filter  it  through  a  common  filter,  or  through  fine 
glass  wool  directly  into  the  glass  bottle. 

B.  Glycerine-jelly.  At  the  present  time  glycerine  is  being 
replaced  by  a  fluid  that  contains  glycerine  *  but  which  is  used 
warm  and  stiffens  on  becoming  cold, —  the  so-called  glycerine- 
jelly.  We  believe  that  this  medium  is  to  be  preferred  in  many 
cases  to  glycerine,  since  it  is  much  more  convenient  to  handle, 
and,  as  we  have  learned  by  careful  experiments,  it  preserves 
many  botanical  objects  in  a  very  superior  way,  always  assuming 
that  good  glycerine-jelly  be  used. 

Kaiser37  gave  a  method  for  preparing  glycerine-jelly  which 
we  can  certify  from  our  own  experience  is  very  satisfactory. 
One  part  by  weight  of  the  finest  French  gelatine  is  soaked  for 
about  two  hours  in  6  parts  by  weight  of  distilled  water.  To 
this  is  added  7  parts  of  chemically  pure  glycerine,  and  to  each 
100  g.  of  the  mixture  add  1  g.  carbolic  acid.  The  mixture 
should  then  be  warmed  with  constant  stirring  for  ten  or  fifteen 
minutes,  till  all  the  flakes  which  were  formed  by  stirring  in  the 
carbolic  acid  have  disappeared.  Finally  filter  while  still  warm, 
through  glass  wool,  which  has  been  previously  washed  and  put 
in  the  funnel  while  still  moist. 

Glycerine-jelly  stiffens  perfectly  in  ordinary  temperature,  and 
so  must  be  warmed  each  time  it  is  used.  For  this  purpose  it 
should  be  kept  in  a  thin  walled  test  tube,  so  that  it  may  be 
warmed  in  a  moment.  Then  a  drop  is  taken  up  by  means  of  a 
glass  rod  and  put  on  the  slide,  the  slide  itself  being  gently 
warmed,  and  the  object  which  has  been  previously  immersed  in 
a  weak  solution  of  glycerine  is  embedded  in  it.  Then  the 
cover-glass  (warmed)  is  put  on  and  the  whole  left  to  cool. 
The  preparation  is  completed  when  it  afterwards  has  been  pro- 
vided with  a  ring  of  varnish  or  cement  around  the  edge  of  the 
cover-glass.38 

Nordstedt39  in  making  glycerine-jelly  dissolves  1  part  of  pure 

87  Raiser  in  Botan.  Centralbl.,  Bd.  1, 1880,  p.  25,  ff.  Vgl.  further  Brandt  in  Zeitsch.,  /,  Mi- 
kroskopie  Bd.  II,  1880,  p.  69,  ff.—  Poulsen,  Botanisk  mikrok.,  p.  43  (Translation  Am.  ed.p. 
67).—  Journal  of  the  Royal  Microscopical  Soc.,  London,  Vol.  Ill,  1880,  p.  502. 

38  Preparations,  Avhosecell  contents  (protoplasm,  chlorophyll,  etc.)  are  to  be  preserved, 
must  be  hardened  as  much  as  possible,  before  they  are  mounted  in  jjlycerine  jelly. 

as  Nordstedt:  Ora  anvandandet  af  gelatinglycerine  vid  undersokning  og  preparering  af 
Desmidieer  (Botaniska  Notiser,  1876,  No.  2).— Poulsen,  I.  c.,  p.  43  (Am.  Trans.,  p.  (37). 


PRESERVING  MEDIA.  221 

gelatine  in  3  parts  boiling  water,  and  adds  4  parts  of  glycerine 
and  (to  prevent  fungus  growth)  a  piece  of  camphor. 

A  third  method  of  making  nnd  using  glycerine-jelly  is  given 
in  the  American  Monthly  Microscopical  Journal40  as  follows  : 
"The  jelly  is  made  by  dissolving  transparent  isinglass  in  suffi- 
cient water  so  that  it  makes  a  stiff  jelly  when  at  the  ordinary 
temperature  of  the  room.  When  the  slides  are  mounted,  add 
one-tenth  as  much  good  glycerine,  and  a  little  solution  of  borax, 
carbolic  acid,  or  camphor  water.  The  mixture  while  hot  should 
be  Avell  filtered  through  washed  linen  or  other  fabric,  as  it  will 
not  go  through  common  filter  paper,  and  the  subsequent  addi- 
tion of  a  little  alcohol  improves  its  working.  Objects,  if  per- 
fectly clean,  may  be  transferred  from  water  directly  to  this 
medium  which  should  be  slightly  warmed  before  using.  The 
cover  is  adjusted*  and  the  slide  put  away  till  a  number  have 
accumulated.  The  cover  should  not  be  pressed  down  too  hard, 
and  a  liberal  amount  of  jelly  used  to  allow  for  shrinkage  in 
drying.  The  slides  may  be  finished  as  soon  as  the  jelly  has  set, 
or  may  be  left  for  several  days.  If  air  bubbles  are  entangled 

«/  •!  C 

they  will  usually  escape  while  drying,  or  they  may  be  driven 
out  by  warming  the  slide  a  little.  When  ready  to  finish  the 
slides,  take  them  to  a  water  cooler  and  let  the  ice-cold  water 
drop  over  them,  while,  with  a  rather  stiff  camel's  hair  brush  all 
the  superfluous  jelly  may  be  washed  away  by  aid  of  the  flow- 
ing water  which  keeps  the  jelly  under  the  cover  hard.  The 
slides  are  then  dried  with  a  towel,  or  cloth  and  finished  with 
a  ring  of  cement." 

For  the  rest,  mixtures  of  glycerine  with  gelatine,  gum  arabic, 
glue  or  other  stiffening  substances  have  been  employed  by  var- 
ious microscopists  for  a  long  time  past.  Schacht's  glycerine 
mixture  consisted  of  1  part  gelatine,  3  parts  water,  4  parts 
glycerine.  Deane  mixed  4  parts  glycerine  with  2  of  water  and 
1  of  gelatine,  the  latter  being  dissolved  in  warm  water  and  the 
glycerine  added.  Klebs  recommended  2  parts  concentrated 
isinglass  solution  with  1  of  glycerine.  Beale  softens  pure  glue 

«o  Vol.  II,  1881,  pp.  4,  5. 

*  The  cover  should  always  be  adjusted  with  the  slide  upon  the  self-centering  turn  table, 
an  apparatus  to  be  described  farther  on,  in  such  a  way  as  to  be  exactly  in  its  center. 
A.  B.  H. 


222  THE  MICROSCOPE  IN  BOTANV. 

in  water,  dissolves  it  in  a  glass  vessel  in  a  water  bath,  adds  a 
like  quantity  of  glycerine  and  filters  through  flannel. 

C.  Canada  Balsam.  In  animal  preparations  this  resin  comes 
into  most  extensive  use  as  a  mounting  medium,  while  in  vegetable 
preparations  it  can  seldom  be  used,  because  it  exercises  a  quite 
too  powerful  clarifying  effect  on  the  cell  walls  of  delicate  sections, 
and  most  vegetable  preparations  containing  water  cannot  bear 
the  previous  dehydrating  necessary  for  mounting  in  Canada  bal- 
sam. But  it  is  principally  applicable  to  the  mounting  of  diatoms , 
hard  seed  coverings,  spores,  siliceous  epidermal  cells,  and 
ground-sections  of  fossil  plants. 

We  should  use  the  best  to  be  found  in  the  market,  having 
the  clearness  of  water,  and  not  too  thick.  Balsam  which  has 
become  too  thick  by  long  keeping  may  be  thinned  by  turpentine 
oil,  chloroform  or  ether.  In  order  to  prevent  evaporation  as 
much  as  possible,  it  should  be  kept  in  wide-necked,  glass-stop- 
pered bottles.  E.  Kaiser  has  recently  put  into  the  market 
Canada  balsam  in  tubes  dissolved  in  spirits  of  turpentine.  It 
is  a  very  limpid,  almost  colorless  fluid  which  is  put  up  in  me- 
tallic tubes  like  oil  colors.  The  balsam  in  this  form  is  very 
handy  to  mannge,  dries  quickly  and  very  seldom  forms  air 
bubbles  in  the  preparation. 

Mounting  in  balsam  is  done  as  follows :  The  object  to  be 
mounted  must  contain  no  trace  of  water,  so  it  must  be  perfectly 
dried  in  a  drying  box  (like  diatoms) ,  or  —  since  all  preparations 
will  not  bear  this  kind  of  drying — the  water  must  be  removed 
by  putting  the  specimen  in  absolute  alcohol  from  which  it  is 
transferred  to  turpentine  oil,  or  oil  of  cloves.  Now  put  a  drop 
of  the  balsam  on  a  slightly  warmed  slide,  take  the  object  from 
the  fluid  in  which  it  is  lying,  let  all  run  off  that  will,  and  then 
put  it  in  the  balsam,  which  should  cover  it  on  all  sides  and  all 
over,  the  slide  being  kept  warm  meanwhile  by  being  held  over 
the  flame  of  a  small  alcohol  lamp  [or  over  the  short  chimney 
of  a  small,  low  kerosene  lamp.  For  all  wanning  purposes  in 
mounting,  such  a  lamp  is  quite  as  good  as  an  alcohol  lamp,  and 
naturally  much  less  expensive  to  keep  going.  I  always  use  one 
under  my  warming  table  in  mounting  with  glycerine-jelly  as 
well  as  with  balsam.  A.  B.  H.]  For  mounting  in  balsam  as 


PRESERVING  MEDIA.  223 

well  as  iii  glycerine-jelly,  it  is  best  to  use  circular  cover-glasses, 
as  there  is  much  less  risk  of  having  air  bubbles  than  when  using 
those  with  corners.  Should  they  occur,  however,  under  the 
cover-glass,  they  may  be  removed  by  leaving  the  slide  in  a  warm 
place  for  a  considerable  time,  in  an  inclined  position,  as  on  the 
iron  plate  of  a  stove.  When  the  preparation  is  several  days 
old  the  balsam  will  become  hard,  and  all  which  has  exuded  about 
the  edge  of  the  glass  may  be  scraped  away  with  a  knife,  and 
the  last  traces  of  it  removed  with  a  cloth,  and  a  little  alcohol, 
or  oil  of  turpentine.  It  is  not  absolutely  necessary  to  put  a 
varnish  ring  around  the  preparation  :  however,  many  microscop- 
ists  take  pains  to  do  it. 

[As  a  substitute  for  Canada  balsam  and  other  solutions  of 
resinous  gums,  used  as  mounting  fluids,  I  have  for  many  years 
employed  with  great  satisfaction  a  fluid  made  after  the  following 
formula. 

(1)  Gum  mastic  38  g.  dissolved  in  55  cc.  of  chloroform. 

(2)  Gum  dammar  38  g.  dissolved  in  55  cc.  spirit  of  terebinth. 
Mix  the  two  solutions  and  filter. 

It  may  be  employed  not  only  as  a  mounting  medium,*  but  also 
as  a  temporary  cement  for  inclosing  glycerine  mounts  with  which 
fluid  it  will  not  mingle  at  all  or  run  in  under  the  cover.  After- 
wards stronger  cement  should  be  applied  over  this  to  keep  all 
secure.  A.  B.  H.] 

D.  Chloride  of  Calcium  Solution.  Chloride  of  calcium 
is  the  oldest  preserving  medium  known  for  permanent  botanical 
preparations,  it  having  been  recommended  by  H.  v.  Mohl.41 
It  was  in  earlier  times  almost  the  only  preserving  medium 
known.  According  to  Harting,  Dippel,  Xageli  and  Schwendener 
it  serves  an  excellent  purpose  for  cell-wall  preparations,  but 
not  for  chlorophyll  and  starch  objects  since  these  swell  up  in 
it  and  are  quite  destroyed.  In  recent  times  chloride  of  calcium 
has  mostly  gone  out  of  fashion,  and  indeed  not  without  good 
reason,  for  it  is  not  easy  to  find  a  cement  that  will  hold  it.  It 
is  used  either  in  a  saturated  solution  or  in  different  degrees  of 
dilution  (1  Ca  Cl.2:4  —  8  H2O,  Harting.  1  Ca  C12 :  ^3  H2  O, 

41  Hugo  v.  Mohl,  Mikrographie,  p.  335. 


224  THE  MICROSCOPE  IN  BOTANY. 

Dippel),  adding  to  the  fluid   some  drops  of  chemically  pure 
hydrochloric  acid. 

E.  Other  Preserving  Fluids.  Besides  those  already  men- 
tioned, there  is  a  whole  series  of  mounting  fluids  to  which, 
however,  but  a  passing  word  can  be  given,  partly  on  account 
of  their  doubtful  value  and  partly  because  they  are  suitable 
for  but  comparatively  few  preparations. 

(a)  /Sugar  water,  with  a  little  corrosive  sublimate  added  to 
prevent   the    growth  of  fungi,    is,    according   to   Nageli    and 
Schwendener,  suitable  for  all  such   objects   as  are   too   much 
changed  by  glycerine  and  calcium  chloride. 

(b)  Solution  of  Corrosive   Sublimate.     Of  this,    numerous 
compounds  have  been  made  and  recommended,  first,  that   of 
Goadby  (Goadby's  Fluid)  : 

Sodium  chloride  120.00  g. 

Alum  60  00  " 

Corrosive  sublimate  0.25  " 

Boiling  distilled  water  2.33    1. 

Pacini  modifies  the  mixture  as  follows  : 

i  ii 

Corrosive  sublimate  1  part  Corrosive  sublimate  1  part 

Common  salt  2     "  Acetic  acid  2    " 

Glycerine  13     "  Glycerine  43    " 

Distilled  water  113    "  Distilled  water  275    " 

The  mixture  is  left  to  stand  for  two  months,  and  is  then  di- 
luted one  part  thereof  with  three  of  distilled  water  and  filtered. 
The  botanist,  however,  finds  little  use  for  compositions  of  cor- 
rosive sublimate. 

(c)  Creosote  Mixture.     Harting  recommends  for  some  prepa- 
rations an  aqueous  solution  of  creosote,  consisting  of  a  saturated 
solution  of  creosote  in  20  parts  of  water,  to  which  1  part  of  30 
per  cent  alcohol  is  added.     Beale  gives  the  following  formula 
for  a  complicated  mixture.     Mix  180  g.  methyl  alcohol  with 
11  g.  creosote,  then  add  enough  pulverized  chalk  to  form  a  thick 
pulp ;  then,  in  a  mortar,  slowly  add  with  constant  trituration, 
1920  g.  water  and  put  in  a  few  pieces  of  camphor.     After  two 
or  three  weeks,  filter  and  keep  the  filtrate  in  a  well  closed  glass 
bottle.     It  is  a  good  preservative  for  desmids. 


PRESERVING  MEDIA.  225 

(d)  Topping's  Fluid  consists  of  one  part  absolute  alcohol  and 
five  parts  water,  or  in  place  of  the  water,  4  parts  water  and  1 
part  acetate  of  alum.     An  equal  volume  of  glycerine  is  added 
to  the  mixture  and  should  be  used  principally  in   preserving 
objects  stained  with  carmine. 

(e)  Potassium  Acetate.     This  salt  was  first  recommended  by 
Sanio,42  and  was  afterwards  designated  as  a   good  preserving 
medium  by  Dippel.     It  preserves  the  chlorophyll  beautifully 
without  a  sign  of  shrinking  of  the  cell  wall.     Use  a  saturated 
solution  in  distilled  water.     It  is  employed  as  a  preservative  for 
bacteria  which  have  been  stained  with  aniline.43 

(/)  Preserving  Medium  for  Algce.  For  preserving  conferva, 
and  related  forms,  including  desmitls  (fresh-water  algae),  P. 
Petit44  employs  the  following  composition  which  is  filtered  after 
solution  : 

Camphor  water  50  00  £. 

Distilled  water  50.00  " 

Glacial  acetic  acid  00.50  " 

Crystal  copper  chloride  00.20  " 

"     "     copper  nitrate  00.20  "    * 

(g)  [King's  Fluid  for  preserving  Marine  Algaz.  Rev.  J.  D. 
King  finds  the  following  composition  entirely  satisfactory  for 
mounting  and  preserving  microscopic  specimens  of  marine 
algae.  Powdered  alum  62.20  g.,  corrosive  sublimate  0.258  g., 
dissolved  in  2.27  1.  of  pure  sea  water  and  filtered.  For  several 
years  past  I  have  made  much  use  of  a  mixture  of  sea  water  and 
glycerine  for  this  purpose.  It  mny  be  mixed  in  various  pro- 
portions, though  5  parts  of  the  former  to  1  of  the  latter,  will 
be  found  to  do  well  in  most  cases.  It  is  a  good  plan  to  filter 
the  sea  water  at  first  and  then  let  it  stand  tightly  corked  in  a 
glass  bottle  for  several  months,  then  filter  again  and  mix. 
A.  B.H.] 


«  Sanio  in  Botan.  Zeitg.  18G3.  p.  359. 

43  Poulsen,  I.  c.,  p.  -U  (Translation,  p.  69). 

44  Brebissonia,  Jahrg.  Ill,  1880,  p.  92.— See  also  Cornu  et  Rivet,  Des  preparations  mi- 
croscopiques,  Paris,  1*72.—  Concerning  a  peculiar  method  of  preparing  for  examination 
marine  algae,  which  have  been  once  dried,  see  C.  F.  Jones  in  Northern  Microscopist,  Vol.  I,  p. 
54-50.   Also  Jour.  Hoy.  Micfosoop.  Soc.,  London,  Vol.  1.,  Series  II.  1881,  p.  530. 

*  Mr.  G.  W.  Morehouse  adds  to  this  100  g.  strong  glycerine,  or  if  the  specilic  gravity  is  too 
high,  less  glycerine.    See  Am.  Monthly  Microscopical  Journal,  Dec.,  1883,  p.  234.    A.  li.  H. 

15 


226  THE   MICROSCOPE   IN  BOTANY*. 

(h)  Monobrom-Napldhaline ,  according  to  the  recent  experi- 
ments of  Abbe  and  Dippel,  is  a  suitable  mounting  medium  for 
objects  which  are  to  be  examined  with  very  high  powers,  as,  for 
example,  diatoms  used  as  test  objects. 

(/)  [tityrax  and  Liquid  Amber.  Dr.  H.  Van  Heurck*  has 
published  an  account  of  his  very  satisfactory  experience  with 
these  resins,  Liquidamber  orientalis  Mill,  and  Liquidamber 
styraciflua  L.  as  a  mounting  medium  for  diatoms,  and  other  such 
objects  requiring  a  medium  of  a  high  index  of  refraction.  In 
this  case  it  is  1.65,  Canada  balsam,  which  this  is  intended  to 
take  the  place  of,  being  1.53.  The  commercial  article  is  first 
dissolved  in  chloroform  and  filtered  to  purify  it  from  the  gran- 
ular substances  which  it  contains, —  and  the  solution  thus  ob- 
tained is  used  in  the  same  manner  as  a  like  solution  of  Canada 
balsam.  It  will  not  form  bubbles  of  air  in  heating.  It  is  re- 
commended to  expose  the  liquid  amber  in  a  thin  layer  to  the 
direct  light  of  the  sun  for  several  weeks.  This  will  cause  it  to 
discharge  all  of  its  water  and  most  of  its  color.  It  becomes 
hard  and  then  may  be  dissolved  as  before  directed  in  chloroform. 
It  may  also  be  dissolved  in  benzine  or  a  mixture  of  benzine  and 
absolute  alcohol.  A.  B.  H.]f 

(&)  Colored  Mounting  Fluid.  A  complicated  preserving  me- 
dium, especially  adapted  for  mounting  starch  grains,  has  recently 
been  given  by  Seiler.45  He  describes  its  preparation  and  use 
as  follows  : 

"  It  is  necessary  first  to  have  some  aniline  blue  staining  fluid 
which  we  make  after  the  formula  given  by  Beale  : 

Soluble  aniline  blue     0.032  g. 
Distilled  water  31.  cc. 

Alcohol  25  drops. 

A  mixture  is  made  of  equal  parts  of  glycerine  and  water,  say 
15  cc.  each,  to  which  are  added  2  or  3  drops  acetic  acid.  To  this 
mixture  of  slightly  acidulated  dilute  glycerine  is  added  the 

*  Bull,  de  la  Soc.  Beige  de  Microscopic.  Quoted  in  Am.  Month.  Micro.  Journal,  Apr., 
1884,  p.  69,  70. 

I  Prof.  11.  lj.  Smith  has  discovered  two  media  for  the  same  purpose,  the  nature  of  which 
he  has  not  yet  chosen  to  make  public,  which  have  an  index  of  refraction  respectively  of 
2.00  and  2.25.  Sec.  J.  D.  Cox.  in  Am.  Monthly  Micros.  Journal,  Apr.,  1884,  p.  71. 

«5  Seller's  Compend.  of  Micro.  Technology,  Phil.,  1881,  p.  13, /. 


PRESERVING  MEDIA.  227 

aniline  blue  staining  fluid  until  the  whole  mixture  is  of  a  decided 
blue  color.  A  drop  of  this  mixture  is  placed  on  a  glass  slide 
and  some  of  the  starch  to  be  mounted  is  dusted  over  the  top. 
The  dusting  can  be  done  to  the  very  best  advantage  by  touching 
the  starch  with  a  camel's  hair  brush  and  then  slightly  shaking 
the  brush  over  the  drop  of  colored  glycerine.  The  starch  soon 
sinks  to  the  bottom  of  the  mixture  and  the  cover  is  applied. 
With  this  method  of  introducing  the  starch,  air  bubbles  are 
avoided.  The  cover  is  pressed  down  quite  firmly  upon  the  slide 
and  the  excess  of  glycerine  carefully  removed.  The  slide  is 
then  transferred  to  the  turn  table  and  a  thin  layer  of  dammar  or 
balsam  in  benzole  placed  around  the  border  of  the  cover.  This 
soon  hardens,  and  in  a  day  or  two  the  slide  may  be  finished  with 
a  ring  of  white  zinc,  Brunswick  black  or  other  cement.  The 
effect  is  this  :  the  grains  themselves  have  not  taken  the  staining 
in  the  least,  neither  will  they  ever  take  it ;  they  retain  their 
natural  appearance  surrounded  everywhere  by  the  blue  glycerine 
and  the  effect  is  most  beautiful." 

(?)  [Mr.  Karl  Castelhun  of  Newburyport,  finds  the  following 
very  satisfactory  for  preserving  sections  of  lichens  : 

Glycerine    7  parts 
Alcohol       1     " 
Water         6      "      ] 

(m)  [Strong  carbolic  acid  is  highly  recommended  for 
preserving  vegetable  tissue.  It  should  be  used  with  only 
so  much  water  added  as  will  -keep  it  from  crystallizing. 
"  One  great  advantage  of  its  use  is  found  in  the  readiness 
with  which  it  penetrates  a  specimen  and  mixes  with  the 
fluids  used  in  mounting,  such  as  water,  glycerine  and  Canada 
balsam.*] 

(n)  [Preservative  for  Fungi.  Thoroughly  mix  1.1341.  white 
wine  vinegar  with  127.5  g.  common  salt  and  141. 7  g.  pulverized 
alum  and  keep  in  a  wide-mouthed  glass  jar.  Put  the  fresh 
specimens  of  fungus  into  it.  From  time  to  time  the  liquid  may 
be  strained  to  take  out  impurities. f] 

*  Wm.  J.  Pow.  in  Am.  Month.  Micro.  Journal,  Jan.,  1883.  p.  8. 

t  Mary  E.  Banning  in  Bulletin  Torrey  Botan.  Club,  Dec.,  ISS2,  p.  153. 


228  THE  MICROSCOPE  IN  BOTANY. 

(o)  [  Wickersheimer's  fluid  for  preserving  algag,  lichens, 
fungi,  etc.,  preserves  the  color  of  most  delicate  structures  quite 

perfectly. 

Alum  100  g. 

Common  salt  25  " 

Saltpeter  12  " 

Carbonate  of  potash  60  " 

White  arsenic  20  " 

Dissolve  by  boiling  in  3000  cc.  of  water.  Cool  and  filter  and 
add  1550  g.  glycerine  and  300  g.  methyl  alcohol  (wood  spir- 
its).*] 

[This  fluid  is  an  excellent  preservative  for  all  kinds  of  land 
plants  ;  and  plants  which  ordinarily  become  stiff  and  brittle  by 
drying  will  always  retain  their  natural  flexibility  if,  previously 
to  drying  they  are  immersed  for  a  little  time  in  this  fluid,  till 
they  become  saturated  with  it.  A.  B.  H.] 

Besides  the  preserving  and  mounting  fluids  here  mentioned, 
there  are  a  large  number  of  others,  formulae  for  which  are 
scattered  through  the  different  works.  We  have  not  included 
them  in  this  work  because  the  greater  part  of  them  have  been 
used  only  by  their  inventors,  their  usefulness  not  having  been 
tested  by  others. 

VIII.     MOUNTING  THE  PREPARATION  IN 
PRESERVING  FLUID. 

"We  have  already  pointed  out  how  to  proceed  in  putting  the 
specimen  into  a  preserving  medium  such  as  glycerine-jelly  and 
Canada  balsam.  This  is  not  usually  attended  with  much  diffi- 
culty and  the  beginner  soon  learns  to  do  it  successfully  after  a 
few  experiments. 

It  is  otherwise  in  mounting  with  fluids.  In  doing  this  we 
have  carefully  to  measure  out  the  exact  quantity  required  to  fill 
the  space  between  the  glass  cover  and  the  slide,  and  then  we 
must  hermetically  seal  up  this  fluid  in  which  the  preparation  is 
immersed,  of  the  method  of  doing  which  we  shall  speak  here- 
after. 

*  Furnished  me  by  Dr.  L.  Schouey  of  New  York  City.    A.  B.  H.] 


MOUNTING  THE  PREPARATION  IN  PRESERVING  FLUID.     229 

The  little  knack  by  which  we  may  take  out  each  time  very 
nearly  the  quantity  of  fluid  we  need  has  already  been  indicated 
in  the  case  of  glycerine  .(see  p.  219).  The  fluid  may  be  pre- 
vented from  running  out  about  the  edges  of  the  cover-glass  by 
putting  a  thin  rim  of  varnish,  hereafter  to  be  described,  upon 
the  slide  which  is  somewhat  smaller  than  the  cover-glass  itself. 
Schacht,  who  invented  this  process,  drew  two  varnish  ledges  on 
the  slide  corresponding  to  the  two  opposite  sides  of  the  square 
cover-glass ;  Dippel46,  three  which  formed  a  square  with  one 
open  side,  and  Nageli47  added  still  a  fourth  thus  closing  np  the 
rim  of  varnish.  Each  "of  these  authors  held  his  method  to  be 
the  best.  We  leave  it  to  the  experienced  worker  to  decide  for 
himself  between  them.  The  round  cells  made  by  means  of  the 
turn  table  are  very  pretty,  but  one  must  naturally  use  the  round 
cover- glasses  with  them. 

Whether  or  not  the  slide  be  provided  with  a  foundation  rin<r, 
the  process  of  putting  the  specimen  in  the  fluid  is  the  same. 
The  slide  is  first  breathed  upon  so  that  the  fluid  will  readily 
adhere  to  it  and  then  a  sufficient  quantity  is  laid  on,  and  the 
section,  which  has  been  previously  freed  from  air,  is  put  into  it. 
The  slide  is  put  under  the  mounting  microscope  and  by  means 
of  a  couple  of  needles  the  object  is  carefully  arranged.  In 
doing  this  it  is  well  to  press  the  section  down  gently  till  it  ad- 
heres to  the  surface  of  the  slide,  so  that  in  putting  on  the  cover- 
glass  one  would  run  no  risk  of  pushing  it  out  of  place.  [To 
avoid  this  danger  and  the  consequent  trouble  caused  by  thrust- 
ing the  preparation  from  its  place,  Rev.  J.  D.  King  recommends 
the  following  method  where  the  preparation  is  to  be  mounted 
in  glycerine  or  any  of  its  compounds.  Put  a  thin  film  of  gly- 
cerine-jelly on  the  slide  where  the  object  is  to  lie,  and  with  the 
warm  slide  and  the  melted  jelly  arrange  the  preparation  where 
it  is  wanted.  Let  the  jelly  cool  and  stiffen,  then  put  on  the 
glycerine,  remove  all  the  air  bubbles  under  the  mounting  mi- 
croscope, put  on  the  cover  and  press  it  down  expelling  all  the 
superfluous  glycerine,  seal  and  cement  in  the  usual  way.  Then 
to  render  the  fluid  in  which  the  preparation  is  mounted  perfectly 

<6  Di|>pel.  1.  c..  Pxl.  I.  p.  474. 

*'  Nageli  u.  Schwdudener,  1.  c.,  p.  297. 


230  THE   MICROSCOPE   IN  BOTANY. 

homogeneous,  warm  the  slide  gently  till  the  glycerine-jelly 
film  is  melted,  when  it  will  mingle  freely  and  perfectly  with  the 
other  fluid.  This  process  is  especially  convenient  where  one 
wishes  to  arrange  several  small  sections  under  one  cover-glass. 
A.  B.  H.] 

The  following  manipulation,  putting  on  the  '  cover-glass, 
must  first  be  taught  to  beginners  who  often  fail.  If  angular 
covers  are  used,  he  should  take  them  up  in  the  forceps  by 
one  corner,  and  having  breathed  upon  the  side  which  will  come 
next  to  the  glycerine  or  other  fluid,  he  puts  down  the  edge 
opposite  to  the  forceps.  Now  he  lowers  the  forceps  till  the  drop 
of  mounting  fluid  touches  the  middle  of  the  glass.  Now,  when 
the  skilful  manipulator  suddenly  lets  go  of  the  cover-glass  it 
falls  into  its  place  so  that  its  edges  lie  parallel  with  those  of  the 
slide,  and  the  mounting  fluid  is  evenly  spread  out  between  the 
two  glasses  and  there  are  no  air  bubbles.  If  we  use  slides 
which  have  a  ring  or  square  of  cement  already  laid  on  them  and 
put  the  edge  of  the  cover-glass  upon  that  while  it  is  yet  sticky, 
it  will  be  fixed  at  once  in  its  place,  which  is  a  matter  of  great 
importance  in  the  subsequent  hermetical  sealing  of  the  cell. 

The  cover  being  now  successfully  laid  on,  we  examine  the 
object  with  a  magnifying  glass  or  the  preparing  microscope,  to 
bee  if  it  is  still  in  its  place  in  the  middle  of  the  cover-glass,  or 
has  been  pushed  aside.  In  the  latter  case  (if  we  have  not 
mounted  the  object  in  a  closed  cement  ring  as  above  described), 
we  may  replace  it  again  by  means  of  a  common  hair  held  be- 
tween the  thumb  and  finger  and  thrust  in  between  the  cover- 
glass  and  slide,  or  by  means  of  a  very  fine  glass  thread  which 
one  may  make  by  drawing  out  a  piece  of  glass  tube  over  the 
flame. 

Very  delicate  preparations,  as,  for  example,  sections  of  the 
very  young  parts  of  flowers,  "growing  points,"  etc.,  are  so 
delicate  that  the  weight  of  the  cover-glass  will  quite  destroy 
them  especially  when  to  this  is  added  a  little  pressure  by  the 
drying  of  the  cement  ring  which  holds  the  cover.  There  are 
several  ways  of  preventing  this.  One  proposed  by  Purkinje48 

48  Purkinje  in  Wagner's  Handbuch  der  Physiologie,  Artikel  Mikroskop. 


MOUNTING  THE  PREPARATION  IN  PRESERVING  FLUID.     231 

and  Hugo  v.  Mohl49  consists  of  laying  small  wax  balls  between 
the  cover-glass,  which  by  gentle  pressure  on  the  latter  can  be 
reduced  to  the  exact  thickness  of  the  preparation.  This  method 
is  in  fact  a  very  good  one.  In  extraordinarily  thin  and  deli- 
cate sections  small  pieces  of  the  fibres  of  glass  wool,  or  of  the 
hair  from  the  head,  may  be  substituted  for  the  wax  balls  to  give 
the  cover-glass  the  right  position. 

But,  on  the  other  h.-ind,  if  the  preparation  is  very  thick,  a  little 
trough  or  cell  about  the  height  of  the  thickness  of  the  section 
must  be  built  up  upon  the  slide,  in  which  it  may  lie.  For  this 
purpose  we  build  up  a  wall  of  varnish  or  shellac,  of  the  size 
and  shape  of  the  cover-glass,  by  putting  on  one  layer  after  an- 
other, letting  each  layer  dry  before  adding  another.  AVe  may 
also  use  wax  instead  of  varnish,  and  then  our  cell  will  soon  bo 
done  for  that  rapidly  stiffens.  But  the  wax  in  thick  layers  is 
always  brittle  and  will  not  without  injury  bear  sudden  changes 
of  temperature.  On  this  account  the  cement  Cells  are  greatly 
to  be  preferred. 

[Making  cement  cells  by  the  use  of  the  self-centering  (or  any 
other)  turn  table,  is  a  matter  of  very  little  difficulty,  but  of  the 
greatest  importance  to  the  microscopist.  For  all  but  the  very 
deepest  cells  they  answer  the  purpose  perfectly,  and  for  these 
metallic  or  glass  rings  may  be  used.] 

[The  strongest  cement  is  the  best  for  cells.  Usually  some 
solution  or  compound  of  shellac  is  preferred,  directions  for 
making  which  will  be  found  below.  Probably  nothing  better 
of  this  kind  can  be  had  than  King's  amber  cement,  or  white 
cement.  The  slide  is  laid  upon  the  turn  table  and  concentrically 
clamped.  A  small  hair  pencil  is  dipped  in  the  cement  and  while 
the  turn  table  is  being  somewhat  rapidly  rotated  the  brush  is 
carefully  brought  down  upon  the  rotating  slide  so  as  to  draw  a 
circular  band  of  cement  3  or  4  mm.  wide  upon  it  in  such  a 
position  that,  when  the  cover-glass  is  put  on,  its  edge  will  come 
about  in  the  middle  of  the  band.] 

[There  are  two  ways  of  completing  the  circular  wall  of  the 
cement  cell.  The  one  is  to  lay  on  the  first  coat  of  cement  so 
carefully  that  the  circle  will  have  exactly  the  diameter  and 

<9  H.  v.  Mohl,  Mikrographie,  p.  328,  ff. 


232  THE  MICROSCOPE   IN  BOTANY. 

breadth  required  in  the  cell.  Let  this  thoroughly  dry.  Then 
put  on  another  coat  very  carefully,  exactly  on  the  top  of  the 
first.  Let  it  dry  and  again  repeat  the  operation  till  the  ring  is 
built  up  high  enough.  When  all  is  done  and  the  last  layer  is 
quite  dry  and  hard,  bring  the  top  of  the  ring  to  a  flat  and  even 
surface  with  a  smooth  file,  or  upon  a  stone.  It  then  may  be 
laid  away  till  wanted.] 

[The  other,  and  perhaps  the  preferable  because  the  more  rapid, 
way  of  making  cement  cells,  is  to  lay  on  a  considerable  quan- 
tity of  the  cement  at  the  first,  making  the  inner  edge  of  the 
circle  come  as  near  as  possible  to  the  position  where  it  is  wanted, 
but  permitting  the  outer  edge  to  spread  out  wider  than  the  ring 
is  to  be  when  finished,  making  it  6  or  7  mm.  broad  if  necessary, 
and  lay  on  all  the  cement  needful  to  finish  the  cell.  Then  with 
the  point  of  a  knife  applied  to  the  slide  at  the  outer  edge  of  the 
cement  ring,  while  the  turn  table  is  rapidly  rotating,  turn  the 
edge  of  the  ring  slowly  inward  narrowing  the  band  and  heaping 
up  the  cement  until  the  desired  height  and  breadth  of  the  ring 
is  attained.  If  the  inner  edge  of  the  ring  should  need  to  be 
turned  and  smoothed  a  little,  it  may  be  done  in  the  same  way, 
but  it  would  not  be  best  to  move  it  very  far,  for  unless  the  work 
is  very  nicely  done  traces  of  the  cement  will  be  found  on  the 
slide  at  the  bottom  of  the  cell.  If  this  should  happen,  the  best 
way  to  clean  it  is  to  let  it  dry  thoroughly,  put  it  again  on  the 
tuin  table,  rapidly  rotate  it,  and  with  the  smooth  point  of  the 
knife  turn  off  or  scrape  up  the  adhering  traces  of  cement.  The 
particles  may  then  be  removed  with  a  dry  cloth  or  camel's-hair 
brush.  The  top  of  the  ring,  when  perfectly  dry,  should  be  made 
smooth  and  flat  as  in  the  other  case.  A.  B.  H.] 

But  if  we  wish  to  make  a  durable  trough  quickly,  we  shall 
have  recourse  to  the  so-called  glass  cell.  It  is  a  perforated  glass 
plate,  represented  in  Fig.  102,  I,  and  is  prepared  from  glass 
0.5  to  1.0  mm.  thick,  rough  ground  on  the  underside,  and  thor- 
oughly cemented  to  the  slide.  [Marine  glue  is  excellent  for 
this,  perhaps  the  best,  but  the  cements  mentioned  above  will 
answer  all  purposes.]  One  can  make  glass  cells  for  himself 
without  much  trouble.  Taking  a  glass  tube  of  about  12  mm. 
interior  diameter  and  a  thickness  of  wall  of  about  3  mm.,  have 


MOUNTING  THE  PREPARATION  IN  PRESERVING  FLUID.     233 
i 

it  sawed  up,  at  a  glass  grinder's  into  rings  .5  to  1.0  mm.  thick, 
Fig.  102,  II.  Then  fasten  them  upon  a  glass  plate  with  Can- 
ada balsam  in  much  the  same  way  as  fossil  wood  is  prepared  for 
grinding,  then  with  emery  or  with  turpentine  oil  on  a  whetstone 
grind  it  flat  and  smooth.  Then  turn  it  over,  recement  it,  clean 
away  the  Canada  balsam  and  grind  the  other  side  in  the  same 
way.  A  still  simpler  way  is  to  take  glass  strips  3  mm.  broad, 
1  mm.  thick  and  pf  sufficient  length,  and  by  holding  them  in 
the  flame  of  a  Bunsen  burner,  bend  them  into  the  form  given 
in  Fig.  102,  III,  welding  it  finally  at  a.  The  joint  should  be 
made  in  the  middle  of  the  side  rather  than  at  the  corner. 


O 


A 

FIG.  102. 

In  mounting  large  specimens  in  shellac,  wax,  or  glass  cells 
the  process  is  as  follows.  The  cell  is  filled  full  of  the  mount- 
ing fluid,  for  example  glycerine,  and  the  specimen  carefully  laid 
in.  When  the  cover  is  laid  on,  it  should  be  fixed  at  one  corner 
with  a  small  drop  of  wax  or  shellac,  which  should  be  allowed 
to  stiffen  or  harden  as  the  case  may  be.  But  if  some  of  the 
fluid  has  run  out  and  got  on  the  cover-glass  it  must  be  carefully 
removed,  a  matter  sometimes  of  no  little  difficulty  and  labor. 
The  first  step  toward  cementing  the  cover-glass  should  be  taken 
only  when  it  and  the  upper  surface  of  the  cell  are  perfectly  cleau 
and  dry. 

It  maybe  mentioned  in  passing,  that  this  method  of  mounting 
especially  commends  itself  for  those  slippery  algae  which,  when 
we  undertake  to  mount  them  without  a  cell,  directly  we  put  on 
a  cover-glass  slip  out  from  under  its  edge.  In  this  way  only 
have  we  succeeded  in  mounting  the  gelatinous  fresh-water  alga 
Balrachospernum  monili/orme,  after  all  other  attempts  to  con- 
fine it  had  failed. 


234  THE   MICROSCOPE  IN  BOTANY. 


IX.     CEMENTING  AND  FINISHING  THE  MOUNT. 

When  the  preparation  has  been  embedded  in  the  preserving 
medium  and  the  cover-glass  laid  on,  the  next  step  is  to  surround 
the  edge  of  it  with  a  border  of  varnish  or  cement,  which  when 
dry  will  fasten  it  to  the  slide,  solidly  and  permanently,  hermet- 
ically sealing  up  the  preparation  (see  Fig.  100,  I,  II).  Before, 
however,  we  describe  this  process,  we  should  become  acquainted 
with  the  nature  of  the  cements  or  varnishes  used  in  it. 


1.    CEMENTS. 

1.  Wax.     This  is  used  in  the  form  of  a  little  wax  candle,  the 
wick  of  which  is  lighted  and  then,  after  a  moment,  when  the 
wax  is  melted  around  it,  we  may  dip  a  camel's -hair  brush  into 
it  and  immediately  draw  the  wax  rim  on  the  slide  around  the 
cover-glass. 

2.  Asphalt  Varnish  (Brunswick  Black).    This  consists  of  a 
solution  of  asphalt  in  like  parts  of  turpentine  and  linseed  oil. 
It  can  be  had  in  any  drug  shop.     For  microscopical  purposes 
the  best  only  should  be  used.     Really   good    asphalt  varnish 
renders  the  very  best  service  in  cementing  microscopical  pre- 
parations notwithstanding  Frey's  assertion  to  the  contrary.     If 
it  gets  too  stiff  it  may  be  thinned  with  oil  of  turpentine. 

3.  Mastic  Varnish.    I  am  not  acquainted  with  the  composi- 
tion of  this  varnish.     The  dissolving  medium  is  alcohol.     It  was 
first  recommended  for  our  purposes  by  Schacht.     The  mieros- 
copist's  varnish  of  E.  Kaiser  appears  to  be  like  it,  which  from 
my  own  experience  I  can  recommend.     Both  kinds  when  they 
become  too  stiff  may  be  thinned  with  absolute  alcohol.50 

4.  Shellac  and  Sealing-wax  Cements.     Dr.  O.  E.  R.  Zim- 
mermann  has  kindly  furnished  me  with  the  formula  of  a  very 
useful  shellac  cement.     "Dissolve  good  brown  shellac  in  abso- 
lute alcohol,  add  aniline  green  and  filter.     The  filtrate  should 
now  be  allowed  to  stand  protected  from  dust  near  a  stove  in  a, 


6°  Frey,  I.  c.,  p.  H3.-Dippel,  I.  c.}  Bd.  I,  p.  473. 


CEMENTING  AND  FINISHING  THE  MOUNT.  235 

wide-necked  glass  bottle  till  it  has  become  so  thick  that  it  will 
not  run  when  put  on  a  slide  with  a  hair  pencil,  but  makes  a 
sharply  defined  outline.  This  varnish  never  cracks  off.  When 
perfectly  dry  and  subjected  to  frequent  changes  of  temperature, 
isolated  wrinkles  will  sometimes  come  in  it,  but  it  never  loosens 
up  so  as  to  injure  the  object." 

Thiersch51  has  given  a  formula  for  making  a  thin  shellac  cem- 
ent which  can  be  used  with  balsam  mounts.  Thick  brown  shel- 
lac cement  (prepared  by  the  solution  of  shellac  in  alcohol)  is 
evaporated  to  the  consistency  of  thin  mucilage  and  colored  with 
a  concentrated  solution  of  aniline  blue  or  gamboge  in  absolute 
alcohol.  To  60  g.  add  at  last  2.5  g.  castor  oil,  evaporate  still 
a  little  further  and  keep  in  a  well-closed  bottle.  If  it  becomes 
gradually  too  much  concentrated  it  may  be  thinned  with  a  few 
drops  of  alcohol. 

Poulsen52  gives  a  recipe  for  a  third  kind  of  shellac  cement 
which  he  names  the  Gram-Riitzou  cement:  "50  g.  Canada  bal- 
sam, 50  g.  shellac,  50  g.  absolute  alcohol,  and  100  g.  ether 
are  mixed  and  evaporated  in  the  water  bath  to  a  thick  syrupy 
consistency. 

An  alcoholic  solution  of  sealing  wax  is  often  proposed  in  place 
of  the  shellac  cements,  but  I  have  no  great  confidence  in  it. 

[I  am  indebted  to  the  kindness  of  Rev.  J.  D.  King  for  two 
formulae,  one  of  a  very  excellent  cement  and  the  other  of  a 
"finish"  which  for  its  cementing  power  and  its  good  appearance 
can  scarcely  be  equalled,  I  think,  by  anything  yet  offered  in  this 
line.  They  are  both  compounds  of  shellac.] 

[King's  Amber  Cement  is  made  as  follows.  (1)  Dissolve  453 
g.  of  best  bleached  shellac  in  half  a  litre  of  95  per  cent  alcohol. 
(2)  In  another  vessel  dissolve  1  part  gum  mastic  in  two  parts 
alcohol  and  let  it  stand  till  perfectly  clear.  To  the  shellac  so- 
lution; (1)  add  38  g.  of  the  mastic  solution,  (2)  color  with 
"dragon's  blood"  dissolved  in  alcohol  and  filter.  Place  it  in  the 
water  bath  and  stir  frequently  till  it  comes  to  a  boil.  Filter 
through  flannel,  after  which,  if  too  thick,  bring  to  a  right  con- 
sistency by  means  of  strong  alcohol.] 

51  Frey,  1.  c.,  p.  143. 

5-  Poulsen,  Botunisk  Mikrokemi.    Translation,  p.  71. 


236  THE   MICROSCOPE   IN  BOTANY. 

[King's  White  Cement  is  made  in  the  same  way  omitting  only 
the  "dragon's  blood."] 

[King's  Lacquer  Finish.*  (1)  Dissolve  453  g.  Dennison's 
excelsior  sealing  wax  in  one-half  litre  or  more,  it'  necessary,  of 
alcohol.  (2)  Dissolve  1  part  best  bleached  shellac  in  2  parts 
95  per  cent  alcohol.  To  every  38  g.  of  No.  1  add  5  g.  of  No. 
2  and  5  g.  of  Brown's  rubber  cement.  Let  it  stand  two  weeks 
or  more  in  a  warm  place,  stirring  it  occasionally.  If  too  thick 
to  flow  freely  reduce  with  alcohol.] 

[The  color  of  this  finish  will  depend  of  course  upon  the  color 
of  the  sealing  wax  used,  and  one  can  thus  exercise  his  taste  in 
ornamenting  his  slides,  at  the  same  time  that  he  secures  in  the 
best  possible  way  the  permanent  safety  of  his  preparations. 
A.  B.  H.] 

5.  Copal  Varnish  can  be  employed  as  a  cement  in  connection 
with  wax  and  asphalt  varnish.    I  prepare  it  in  this  way.     I  put 
5  g.  of  pulverized  copal  in  a  glass  retort  and  pour  over  it  5  cc. 
each  of  absolute  alcohol  and  oil  of  turpentine  and  1  cc.  of  ether 
and  carefully,  slowly  and  gently  warm  until  the  copal  is  dis- 
solved.    Close  the  vessel,  set  it  off  and  pour  the  clear,  transpar- 
ent varnish  into  a  well  closed  glass-stoppered  bottle. 

6.  Dammar  VarnisJt,  used  in  connection  with  the  foresfoinsr 

o          o 

is  prepared  in  the  following  way.  The  best  coarse  grained 
dammar  gum  should  be  warmed  a  long  time  till  all  the  water  is 
driven  out,  then  pour  over  it  three  times  its  weight  of  turpentine 
oil  and  when  dissolved  decant  the  clear,  colorless  varnish  into 
a  glass-stoppered  bottle. 

7.  Gold-size,  a  cement  much  used  by  the  English  is  pre- 
pared, according  to  Beale,  in  the  following  way.     Twenty-five 
parts  of  linseed  oil  are  boiled  three  hours  with  1  part  vermil- 
lion  and  £  part  umber.     The  clear  liquid  is  then  poured  off  and 
like  parts  of  well  ground  white  lead  and  yellow  ochre  are  slowly 
and  gradually  mixed  in  with  constant  stirring,  further  boiled  and 
finally  turned  off  and  kept  in  a  bottle  for  use. 

*  These  cements  and  finishes  may  be  had  ready  made  of  Rev.  J.  D.  King,  Cottage  City, 
Mass. 


CEMENTING  ANGULAR  COVER-GLASSES.  237 

2.    CEMENTING  ANGULAR  COVER-GLASSES. 

[Inasmuch  as  this  form  of  cover-glass  is  very  little  used  in 
this  country  in  botanical  work,  I  shall  condense  what  the  author 
has  to  say  upon  it  into  as  little  space  as  possible.  A.  B.  H.] 

The  tools  to  be  used  are  small  artists'  hair  pencils  of  the 
form  represented  in  Figs.  103  and  104,  the  flat  one  for  laying 
on  the  cement  band  and  the  smaller  round  one 
for  touching  up  and  finishing  off  the  work,  add- 
ing a  little  of  the  cement  here  and  there.  They 
should  be  kept  scrupulously  clean  and  the  cement 
should  never  be  allowed  to  dry  or  harden  in  them. 
This  may  be  prevented  by  having  little  bottles 
partly  filled  with  the  solvent  of  the  cement  in 
which  the  brush  is  used,  alcohol,  turpentine, 
ether,  etc.  ;  and,  having  a  hole  though  the  cork, 
put  the  handle  of  the  brush  in  it,  letting  the 
brush  dip  into  the  liquid  and  there  to  remain 
when  not  in  use.  The  brushes  may  also  be 
cleaned  after  each  using  with  the  cement  solvent, 
and  left  dry. 

If,  in  placing  the  cover,  a  small  drop  of  the 
mounting  fluid,  glycerine,  etc.,  runs  out  upon      FIGS.  103  &  lot. 
the  slide  it  should  be  cleaned  off  before  cement- 
ing.     A  hair  pencil  saturated  with  oil  of  turpentine  will  do 
this,  and  the  traces  of  turpentine  may  be  washed  away  with 
a  brush  dipped  in  ether. 

When  a  foundation  cell  has  been  made  and  the  cover-glass 
pressed  down  upon  the  soft  cement  in  mounting  so  as  to  stick  fast 
all  around,  the  subsequent  cementing  and  finishing  are  an  easy 
matter.  It  is  only  necessary  theivto  fill  the  flat  brush  with  the 
cement  and  draw  it  slowly  along  the  edge  of  the  cover-glass, 
half  on  that  and  half  on  the  slide,  making  the  layer  not  more 
than  3  or  4  mm.  wide  altogether.  Begin  at  the  corner  and  go 
the  length  of  the  side  at  a  stroke.  So  all  around.  The  cem- 
ent or  varnish  for  the  first  coat  should  be  of  as  thick  a  consist- 
ency as  can  be  conveniently  managed  with  the  brush.  The  slide 
should  be  laid  aside  for  a  day  or  two  for  the  varnish  to  dry ; 


238  THE  MICROSCOPE  IN   BOTANY. 

then  examine  it  with  a  magnifying  glass  to  see  if  it  is  all  tight ; 
if  not,  apply  more  thick  varnish.  If  the  first  coat  is  not  substan- 
tial enough,  apply  a  second  of  a  varnish  of  thinner  consistency, 
making  the  band  extend  a  little  beyond  the  edges  of  the  first 
one.  After  a  fortnight,  even  a  third  coating  may  be  put  on. 

If  no  foundation  cell  has  been  made  and  the  cover-glass  has 
nothing  to  stay  it  but  the  adhesion  of  the  mounting  fluid,  a  drop 
of  thick  varnish  should  be  put  at  each  corner  and  allowed  to 
harden,  then  the  varnish  applied  as  before,  care  being  taken  to 
use  pretty  thick  varnish  so  that  it  will  not  run  in  under  the 
cover  and  spoil  the  specimen.  [If,  with  glycerine  as  a  mount- 
ing fluid,  a  chloroform  or  benzole  solution  of  Canada  balsam  or 
dammar,  or  the  mastic  and  dammar  solution  described  on  p.  223 
be  used  for  a  first  coat,  there  will  be  no  danger  of  its  running 
under  the  cover,  and  one  will  find  it  very  convenient  to  use  one 
or  the  other  of  these  with  all  glycerine  mounts  whether  with 
square  or  circular  glasses.  A.  B.  H.] 

Several  coats  of  the  finishing  cement  should  be  applied  one 
after  the  other  as  they  become  dry. 

Another  method  is  to  make  the  first  layer  of  wax.  The  wick 
of  a  small  wax  candle  is  heated  over  the  flame  till  it  is  thor- 
oughly saturated  with  the  melted  wax  and  then  drawn  carefully 
along  the  edges  of  the  cover-glass.  It  immediately  stiffens 
and  the  cement  may  be  applied  at  once.  I  usually  put  over  a 
thin  layer  of  wax  a  layer  of  copal  varnish  which  very  quickly 
dries,  and  then  over  that  a  third  of  dammar  varnish  which  dries 
very  slowly  but  is  very  durable.  When  it  is  dry  I  put  on  at  in- 
tervals three  layers  of  asphalt.  Balsam  and  gelatine  preparations 
should  have  one  or  two  coats  of  asphalt.  Thiersch's  shellac 
cement  may  be  used  with  the  former,  after  a  previous  layer  of 
Canada  balsam  dissolved  in  chloroform  has  been  applied.  [For 
all  finishing  processes  I  know  of  nothing  better  than  Brown's 
rubber  cement,  or  King's  lacquer  finish,  the  latter  being  the 
stronger  and  therefore  the  better.  The  white  zinc  cement  so 
extensively  used  in  this  country  is  bitterly  complained  of  by 
some  and  highly  recommended  by  others.  There  seems  to  be 
no  way  of  accounting  for  such  marked  differences  of  experience 
and  opinion.  It  has  served  me  well.  A.  B.  H.] 


MOUNTING  WITH  CIRCULAR  COVER-GLASSES.  239 


3.    MOUNTING  WITH  CIRCULAR  COVER-GLASSES. 

The  use  of  circular  cover-glasses  is  very  much  to  be  preferred. 
They  are  easier  to  cement,  and  are  more  secure,  not  having  the 
weak  points,  the  corners  of  the  square  ones,  and  it  requires 
much  less  time  to  cement  them,  and  they  also  have  a  much 
more  elegant  appearance  than  the  angular  ones. 

[Mounting  with  and  cementing  circular  covers  requires  the 
use  of  a  turn-table.  Those  with  a  device  for  self-centering  are 
so  much  better  than  those  without  that  I  would  recommend  no 
one  to  get  any  other.] 

[Two  forms  are  herewith  represented  in  Figs.  105  and  106. 
The  construction  of  them  is  so  obvious  that  a  detailed  descrip- 
tion will  not  be  necessary.  The  circular  brass  plate,  Fig.  105, 


FIG.  105. 

revolves  on  a  central  pivot  smoothly,  and  with  the  least  possible 
friction.  It  is  actuated  by  a  stroke  of  the  hand  along  its  milled 
margin.  The  self-centering  apparatus  consists  of  two  rectan- 
gular jaws  upon  the  upper  surface  of  the  plate  which  are  made 
to  clasp  the  slide  at  its  diagonal  corners.  "\Vheu  the  slide  is 
held  by  these  jaws  it  is  concentric  with  the  plate  and  with  its 
axis  of  motion.  The  jaws  are  moved  toward  each  other  by  a 
spiral  spring  beneath  actuating  parallel  bars  to  the  ends  of  which 
the  jaws  are  attached,  and  are  guided  by  the  screws  which  hold 


240  THE  MICROSCOPE  IN  BOTANY." 

them,  moving  in  the  slots  in  the  plate.  The  jaws  are  opened 
by  pressing  upon  the  other  end  of  either  of  the  bars  with  the 
thumb  beneath  the  plate,  while  the  forefinger  of  the  same  hand 
holds  the  plate  above.  Two  spring  clips  are  provided  for  re- 
finishing  old  slides  which  have  been  mounted  without  centering. 
This  turn-table  is  made  by  Beck  of  London  and  sold  in  this 
country  by  Wm.  Walmsley  and  Co.,  of  Philadelphia.] 

[The  -Bauson  and  Lomb  Optical  Company  make  a  turn-table 
which  is  provided  with  a  hand  rest,  which  when  in  use  lies  down 
in  such  a  way  as  to  project  over  the  table  and  the  slide,  but  not 
touching  either,  giving  the  hand  a  perfectly  steady  support  in 
the  manipulation  of  applying  a  ring  of  cement  to  the  slide.] 


FIG.  106. 

[Fig.  106  represents  another  form  of  the  self-centering  turn- 
table, made  by  Mr.  Zentmayer  of  Philadelphia.  The  plan  for 
centering  the  slide  is  something  quite  new.  The  slide  is  cen- 
tered laterally  by  having  its  opposite  sides  brought  in  contact 
with  two  pins  projecting  from  the  plate.  It  is  centered  longi- 
tudinally by  means  of  a  ring  with  an  oval  inner  edge,  which  is 
fitted  to  the  periphery  of  the  disk  in  such  a  way  that  by  turning 
it,  this  inner  edge  of  the  ring  grasps  the  slide  at  its  diagonally 
opposite  corners.] 

[A  form  of  apparatus  for  holding  and  centering  the  slide,  and 
the  mechanism  for  actuating  it,  which  it  is  believed  has  certain 


SELF-CENTERING  TURN  TABLES. 


241 


marked  advantages  in  manipulation  over  other  forms  of  self- 
centering  turn-tables,  has  been  contrived  by  the  writer  and  is 
represented  in  Fig.  107.  The  general  construction  of  the  ro- 
tating plate,  pivot,  frame,  etc.,  is  the  same  as  in  Fig.  105.  Fig. 
107,  A  and  B^  represents  an  outline  of  the  plate  and  attached 
apparatus  f  the  natural  size.  In  A  the  plate  is  seen  from  above, 
aa  are  two  pins  fixed  in  the  plate  equidistant  from  the  center, 
by  which  the  slide  is  laterally  centered,  bb  are  two  pins  mov- 
able in  the  slots  cc,  and,  having  a  diameter  above  greater  than 


FIG.  107  A. 

that  of  the  slots,  their  shoulders  bear  upon  the  upper  surface  of 
the  plate.  These  pins  center  the  slide  longitudinally.  In  _Z?is 
shown  the  apparatus  on  the  under  side  of  the  plate,  by  which 
the  pins  bb  are  moved  in  clasping,  centering,  and  releasing  the 
slide.  These  two  pins  bb  are  screwed  fast  to  two  short  rods  oo. 
The  rods  are  joined  to  the  long  bent  bars  tr  tr  at  nn  with  a 
hinge  joint.  These  bars  are  bent  at  uu  where  they  work  upon 
pivots  made  fast  to  the  plate.  A  pin  10  or  12  mm.  long  is  in- 
serted in  the  end  of  the  outer  bar  at  x.  The  short  arms  ?v  are 

16 


242 


THE  MICROSCOPE  IN  BOTANY. 


pressed  outward  at  x  by  the  strong  steel  spring  s.  This  action 
brings  the  pins  bb  firmly  against  the  middle  of  the  ends  of  the 
slide,  thus  centering  it  longitudinally  and  holding  it  fast.] 

[By  grasping  the  plate  at  m  between  the  thumb  and  forefinger 
of  the  left  hand,  the  plate  is  held  fast,  while  the  finger  presses 
against  the  pin  at  x  and  moves  back  the  centering  pins  bb  along 
the  slots  cc,  while  the  right  hand  is  left  entirely  free  to  manip- 
ulate the  slide.  An  inward  movement  of  x  equal  to  3  mm. 
will  separate  the  pins  bb  a  distance  of  12  mm.  The  pin  e  pre- 


FlG.  107  B. 

vents  the  centering  pins  bb  from  coming  down  to  the  ends  of 
the  slots  at  cc  when  no  slide  is  on  the  plate.  As  it  is  screwed 
into  the  plate,  it  may  be  removed  if  very  short  slides  are  being 
used.] 

[In  the  use  of  the  self-centering  turn-table  the  object  itself 
must  be  placed  in  the  center  of  the  slide.  When  a  cell  is  used 
and  that  cell  has  already  been  made  by  means  of  the  self-cen- 
tering turn-table,  the  object  can  be  mounted  and  the  cover 
adjusted  by  that..  But  when  no  cell  or  ring  of  cement  has  been 


SELF-CENTERING  TURN-TABLES.  243 

previously  put  on,  which  may  serve  as  a  guide,  there  must  be 
something  to  indicate  the  central  point  of  the  slide.  Two 
things  may  be  done.  We  may  place  the  slide  on  the  turn-table 
in  the  usual  way,  and  as  it  lies  there  self-centered,  the  concen- 
tric rings  cut  in  the  disk  will  be  a  sufficient  guide  for  mounting 
the  object  and  adjusting  the  cover-glass.  Or  we  may  lay  the 
slide  its  poorest  side  up  if  there  is  any  choice,  on  the  turn- 
table, and  with  a  hair  pencil  dipped  in  India  ink  draw  a  circle 
on  it  as  near  as  possible  to  the  size  of  the  cover-glass  to  be 
used.  When  this  is  dry  it  will  serve  as  a  guide  in  placing  the 
object  and  adjusting  the  cover,  on  the  other  side  af  the  slide, 
near  enough  for  all  practical  uses.  When  the  slide  goes  on  the 
turn-table  for  cementing  if  it  is  found  that  the  cover-glass  is 
not  exactly  concentric  with  the  motion  of  the  table  it  should  be 
carefully  and  gently  pushed  over  to  its  true  place  with  a  dis- 
secting needle.  It  may  not  perhaps  need  to  be  moved  half  a 
millimeter,  but  it  should  be  perfectly  centered  if  possible  be- 
fore any  cement  is  applied.  A.  B.  H.] 

Supposing  now  we  have  a  preparation  on  the  slide  mounted 
in  glycerine  and  a  round  cover-glass  laid  on,  the  glycerine  hav- 
ing been  so  carefully  measured  out  that  no  trace  of  it  can  be 
seen  beyond  the  edge  of  the  cover ;  on  three  or  four  points  at 
the  edge  of  the  cover-glass  is  placed  a  drop  of  thick  cement  for 
a  stay,  as  already  described  in  regard  to  the  rectangular  glasses. 
When  these  are  dry  the  slide  is  put  on  the  turn-table  and  clamped 
and  the  cover-glass  centered  on  the  table.  [Our  author  is 
not  speaking  here  of  the  new  self-centering  turn-tables.  With 
those  described  above  it  is  obvious  that  the  cover-glass  must 
be  made  concentric  with  the  turn-table  before  it  is  made  fast 
with  these  temporary  stays  of  cement.  And,  indeed,  if  glyce- 
rine is  the  mounting  fluid,  there  will  be  no  need  of  these  at  all 
if  Canada  balsam  or  the  mounting  fluid  of  gum  mastic  and 
dammar,  described  on  p.  223,  be  used  as  the  first  layer  to 
enclose  the  preparation.  A.  B.  H.] 

We  can  now  proceed  to  apply  the  first  ring  of  cement.  Fill 
a  small  hair  pencil  with  not  too  much  of  the  cement,  which  we 
have  chosen  to  use  (there  should  be  no  drops  of  it  hanging 


244  THE  MICROSCOPE  IN  BOTANY. 

from  the  pencil),  hold  it  in  a  perpendicular  direction  close  over 
the  edge  of  the  cover-glass  and  set  the  turn-table  in  slow  motion. 
Suddenly,  but  gently,  lower  the  pencil  till  it  touches  and  the 
next  moment  raise  it  again.  The  ring  is  done.  On  the  steadi- 
ness and  accuracy  of  this  motion  alone  depends  the  success  of 
the  process,  and  it  can  easily  be  attained.  One  should  be  careful 
not  to  take  too  much  cement  in  his  brush  else  the  ring  will  not 
be  uniform,  particularly  in  breadth.  A  second  and  third  ring, 
each  broader  than  the  preceding,  should  be  laid  upon  the  first. 
If  a  cement  ring  was  laid  upon  the  slide  before  the  object  was 
mounted  and  the  cover-glass  pressed  down  upon  it,  we  shall 
not  need  the  stay  drops  of  cement,  the  glycerine  will  not  be 
likely  to  run  out,  and  the  final  cementing  becomes  a  simple  mat- 
ter. The  breadth  of  the  last  ring  need  not  be  more  than  4mm. 
The  author  has  prepared  many  slides,  which  have  remained  un- 
changed for  years,  whose  last  cement  ring  with  a  diameter  of 
18  mm.  was  not  more  than  2  mm.  broad.  [The  ring  may  be 
made  as  narrow  as  one  pleases,  by  turning  in  the  edge  of  it 
from  the  outside  with  the  point  of  a  knife  as  described  in  the 
paragraph  on  making  cement  cells,  p.  232.] 

[I  am  indebted  to  Rev.  J.  D.  King  for  a  process  of  sealing 
cells  with  heat  that  will  be  found  very  useful.  The  cell  is  made 
of  shellac  cement  or  lacquer  finish  and  completed  as  already 
described.  Before  using,  ring  the  outer  half  of  the  flattened 
top  of  the  cell  slightly  with  shellac  cement.  When  the  object 
is  immersed  in  the  glycerine  or  any  aqueous  mounting  fluid,  put 
on  the  cover,  adjust  it  and  press  it  down  carefully  to  its 
bearings  all  around.  Then  apply  a  spring  clip  which  has  a  gen- 
tle pressure  and  pass  the  slide,  cover  down,  a  few  times  over 
the  flame  of  a  spirit-lamp  till  the  cement  shows  signs  of  melting. 
Remove  the  clip,  press  down  the  cover  again  at  any  point  nec- 
essary and  then  hold  it  under  the  cold-water  faucet  to  wash  off 
the  glycerine  and  cool  it,  after  which  carefully  clean,  and  com- 
plete the  cementing  to  fancy  with  shellac  cement  and  lacquer 
finish.  A.  B.  H.] 


LABELING  AND  CATALOGUING. 


245 


X.  LABELING  AXD  CATALOGUING  THE 
PREPARATIONS. 

A.  The  Label.  Every  preparation  must  be  carefully  labeled 
in  order  to  be  easily  found  and  identified.  The  proper  labels 
have  a  rectangular  form  about  as  shown  in  Fig.  100,  I,  II,  IY, 
or  Fig.  98,  or  as  it  suits  the  taste  of  the  preparator  to  make 
them.  They  are  fastened  on  with  a  thick  solution  of  gum 
arable,  or  better  still  with  a  solution  of  brown  shellac  with 
absolute  alcohol.  With  the  latter,  one  may  cover  the  whole 
upper  surface  of  the  label  and  so  render  the  writing  indestruct- 
ible. In  beginning  a  collection  of  microscopical  preparations 
one  should  choose  different  colors  for  the  labels  and  use  each 
color  for  a  limited  group  of  objects.  For  example  :  white  for 
anatomy  of  the  vegetation  organs  of  the  phanerogams;  blue, 
anatomy  of  flowers ;  green,  vascular  cryptogams ;  red,  algae ; 
etc.,  etc. 

The  writing  on  the  labels,  of  which  every  slide  should  have 
two,  should  include  the  following  points : 

1.  Name  of  plant  from  which  the  preparation  is  made.     For 
example,  Pi-unus  avium. 

2.  The  part  of  the  plant  used  in  the  preparation  (stem). 

3.  The  kind  of  section  (transverse,  radial  or  tangential.) 

4.  The  method  of  preparation  (aniline  staining). 

5.  The  mounting  fluid  (glycerine). 

6.  The  date  of  mounting  (15  V,  84). 

The  points  1-3  should  be  entered  on  the  left  hand  label  and 
4-6  on  the  right : 


Besides  these  labels  it  is  well  to  write  on  the  under  side  of 
the  slide  with  a  diamond  the  catalogue  number  of  the  specimen 
in  order  to  identify  it  in  case  the  left  hand  label  should  get  lost. 


246  THE   MICROSCOPE   IN  BOTANY. 

[B.  The  Catalogue.  Every  collection  of  microscopical  prep- 
arations should  be  carefully  catalogued.  As  between  the  use 
of  a  book  or  a  card  catalogue  I  am  inclined  to  prefer  the  latter ; 
it  seems  to  allow  a  somewhat  freer  classification  of  the  object, 
with  opportunities  for  throwing  out  specimens  that  are  no  longer 
desired,  together  with  the  card  that  represents  them,  without 
defacing  the  catalogue,  as  it  would  with  a  book  by  erasures. 
The  catalogue  should  furnish  a  perfect  index  to  the  collection, 
each  slide  being  represented  by  a  card.] 

[The  classification  of  the  preparations  in  the  cabinet  should 
be  made  in  accordance  with  the  natural  system,  in  the  main  or 
primary  divisions  ;  and,  in  accordance  with  the  parts  of  the  plant 
which  they  represent,  for  the  secondary  division.  For  example  : 
the  primary  divisions  should  distinguish  between  the  phanero- 
gamic and  cryptogamic  plants,  and  of  the  former  between  the 
monocotyledons  and  the  dicotyledons  and  again,  perhaps,  of 
the  latter  of  these  between  woody  and  herbaceous  plants,  and 
between  deciduous  and  coniferous  woods,  etc.  Under  these  the 
secondary  divisions  should  recognize,  and  put  together,  sections 
or  other  preparations,  made  severally  irom  the  roots,  stems, 
leaves,  flowers  and  seeds  of  the  plant.] 

[The  number  of  the  slide  then,  after  this  arrangement,  should 
represent  its  place  in  the  cabinet.  A  Roman  capital  A,  B,  C, 
should  represent  the  compartment  of  the  cabinet  in  which  it  be- 
longs, the  Roman  numerals  I,  II,  III,  etc.,  the  particular  box, 
drawer  or  tray  which  contains  it,  and  the  Arabic  numerals  1,2, 
3,  etc.,  the  number  of  the  slide  in  this  particular  holder.  Thus 
each  slide  would  be  numbered  in  this  way,  B,  XV,  15.  The 
position  of  the  cards  in  their  box  should  answer  exactly  to  that 
of  the  corresponding  slides  in  the  cabinet.] 

[If  an  alphabetical  index  is  desired  in  order  to  get  the  readiest 
possible  access  to  any  slide  representing  any  plant  in  the  col- 
lection, it  may  be  had  in  a  supplementary  card  index  catalogue, 
arranged  alphabetically  according  to  genera  and  species.  Let 
cards  be  printed  with  a  blank  for  the  proper  scientific  name  of 
the  plant  from  which  the  preparation  is  made,  and  ruled  columns 
for  the  five  minor  heads,  under  which  the  slides  are  all  classified, 
as  follows :] 


STORING  PERMANENT  PREPARATIONS, 


247 


SCIENTIFIC   NAME    OP    THE    PLANT. 

ROOTS. 

STEM. 

LEAVES. 

FLOWERS. 

SEEDS. 

^B.  XII.  9 

B.  XX.  6  r 

B.  XV.  23 

B.  XXI.  21. 

B.  XXL  18. 

B.  XXV.  6. 

[Thus  a  single  index  card  could  easily  be  made  to  represent 
at  least  50  slides  if  necessary.] ' 

[What  should  the  catalogue  cards  contain  ?     Various  answers 
have  been  made  to  this.     I  will  indicate  what  seems  to  me  mos  t 
important,  following  mainly  the  plan  proposed  by  Prof.  S.  H. 
Gage*  for  catalogues  of  preparations  of  animal  histology.] 
[1.    The  scientific  name  of  the  plant. 

2.  The  cabinet  number  of  the  preparation. 

3.  The  part  of  the  plant  from  which  the  preparation  is  made. 

4.  The  special  purpose  of  the  preparation.     What  it  is  meant 
to  show. 

5.  The  special  method  of  preparation.     Whether  it  was  pre- 
viously hardened,  softened,  or  cut  in  a  natural  state.     Whether 
mounted  whole,  teased  out  into  its  elementary  cells  or  fibers, 
or  cut  into  sections,  and  if  cut  how,  free-hand,  or  by  microtome. 

6.  The  bleaching  or  clarifying  agent,  if  any,  and  how  long  a 
treatment  was  required. 

7.  The  staining  medium  and  time  required. 


The  mounting,  cementing  and  finishing  media. 
Objectives  to  be  used  in  its  study. 
The  date  of  preparation  and  name  of  preparatory 
General  remarks  including   references   to  literature  and 


8. 

9, 

10. 

11, 

good  figures.     A.  B.  H.] 

XI.     STORING  PERMANENT  PREPARATIONS. 

Finished  preparations  should  be  kept  in  a  box  or  case,  which 
is  so  arranged  that  the  slides  resting  near  each  other  occupy 

*  Prof.  S.  II.  Gage,  Proceed.  Am.  Soc.  Microscopists,  Chicago  Meeting,  1883,  p.  169,^. 


248  THE  MICROSCOPE  IN  BOTANY. 

the  least  possible  space.  Microscopical  cabinets  should  satisfy 
the  following  requirements.  By  shutting  tightly  they  should 
protect  the  preparations  from  dust.  They  should  not  allow  the 
slides  to  move  about,  and  should  permit  them  to  lie  in  an  hori- 
zontal position,  where  they  may  be  most  easily  got  at. 

[For  transporting  slides  special  boxes  should  be  used  and  not 
those  in  which  they  are  usually  contained.  The  principal  op- 
tical firms  in  this  country  offer  an  assortment  of  object  cabinets, 
which  for  convenience  of  arrangement  and  excellence  of  work- 
manship leave  nothing  to  be  desired.  But  they  are  mostly  so 
costly  as  to  be  of  the  nature  of  a  luxury,  and  most  microscop- 


FlG.  108. 


ists  are  obliged  to  satisfy  themselves  with  the  cheaper  forms, 
such  as  wood  or  paper  boxes  with  wooden  racks,  or  construct  a 
cabinet  for  themselves  by  some  contrivance  of  drawers  or  trays 
which  has  the  merit  at  least  of  cheapness  if  not  of  elegance.] 

[Recently  two  adaptations  of  an  old  form  of  object  box  have 
been  made  which  make  them  answer  practical  ends  in  a  very 
satisfactory  way  and  at  a  cost  that  brings  them  within,  the  reach 
of  all.] 

[One,  represented  in  Fig.  108  is  a  26  slide  box  made  of  stiff 
paper  board  with  wooden  racks,  and  a  hinged  and  indexed  cover 
which  when  shut  down  holds  the  slides  securely  in  place,  and 
the  whole  shoves  into  a  paper-board,  cloth-bound  case,  shown 


THE  EXAMINATION  OF  LIVING  ORGANISMS.  249 

beneath  the  box  in  the  illustration,  which  makes  all  secure. 
A  circular  blank  is  shown  at  the  top  wherein  to  write  the  num- 
ber of  the  box  for  cataloguing.  When  the. box  is  placed  on  end 
the  slides  are  horizontal.  These  boxes  may  be  kept  on  shelves 
like  bound  books.] 

[The  other  is  called  "  Pillsbury's  Cabinet,"  and  is  shown  in 
Fig.  101).  It  consists  of  a  polished  cherry  cabinet  containing 
twenty  wooden-racked  slide  boxes,  each  holding  twenty-five 


FIG.  109. 

slides.  In  the  illustration  one  box  is  shown  removed  from  the 
case,  with  its  cover  off  and  some  slides  in  place.  The  top  end 
of  each  box,  as  placed  in  the  cabinet,  is  provided  with  an  index 
and  on  the  bottom  of  the  box  inserted  under  each  slide  is  a  cor- 
responding number.  When  the  boxes  are  in  place  the  slides  lie 
horizontal  and  a  list  of  all  the  slides  which  they  contain  is  spread 
out  before  the  eye.  A  cabinet  of  this  kind  capable  of  storing 
500  slides  is  offered  for  $3.50.  J.  W.  Queen  and  Co.,  Phila., 
make  these  two  forms  of  slide  holder.  A.  B.  H.] 

XII.      THE  EXAMINATION  OF  LIVING  ORGANISMS. 

There  are  numerous  objects  which  may  be  examined  under 
the  microscope  in  a  living  state,  as,  for  example,  microscopic 
algae  and  fungi.  Some  of  these  we  might  desire  to  keep  under 
the  microscope  for  a  long  time  in  order  to  observe  their  devel- 


250 


THE  MICROSCOPE  IN  BOTANY. 


opment  or  their  method  of  propagation.  For  this  purpose  a 
simple  slide  and  the  cover-glass  and  the  object  between  are  not 
very  suitable,  for  the  water  quickly  evaporates  from  under 
the  cover-glass  so  that  it  frequently  has  to  be  renewed ;  this 
gradually  increases  the  percentage  of  mineral  substances  held 
in  solution  in  the  water,  and  the  object  is  soon  brought  into 
quite  abnormal  conditions  for  its  life  processes.  Many  contriv- 
ances have  been  invented  for  retarding  or  preventing  the  evap- 
oration of  the  water.  The  older  microscopists  used  a  contrivance 
which  consisted  of  two  brass  rings  one  of  which  screwed  upon 
the  other.  Into  each  was  fitted  a  little  glass  plate,  the  lower 
one  made  somewhat  concave.  The  drop  of  water  with  the 
object  was  put  into  this  and  the  other  glass  screwed  down  tightly 
upon  it.  With  the  older  instruments  of  Schieck  this  apparatus 
had  a  diameter  of  28  mm.  and  a  height  of  9  mm. 


FIG.  110. 

For  a  like  purpose,  a  slide  about  1.5  mm.  thick  with  a  small 
concavity  about  13  mm.  wide  ground  in  it  answers  well.  And 
laterally  a  thick  slide  (about  3.5  mm.)  has  been  used  in  which 
a  ring-like  groove  is  cut  about  3  mm.  deep  and  of  like  breadth. 
[The  "Weber  life  slide"  sold  by  opticians  in  this  country  an- 
swers the  same  purpose  even  better.  The  bottom  of  the  cell 
ground  in  this  slide  is  convex.  A.  B.  H.) 

But  by  far  the  best  method  of  observing  living  objects,  or 
those  under  cultivation,  is  by  means  of  the  hanging  or  sus- 
pended drop.  It  may  be  arranged  as  follows. 

A  ring  of  wax  is  put  on  the  slide  making  a  pretty  deep  cell 


THE  EXAMINATION  OF  LIVING  ORGANISMS.  251 

and  wide  enough  for  the  cover-glass  used.  On  the  cover-glass 
there  is  put  a  drop  of  water  and  the  object  in  that.  It  is  then 
turned  quickly  over  so  that  the  drop  will  hang  suspended  on  the 
tinder  side.  The  cover  is  then  placed  on  the  wax  ring  and  put 
under  the  microscope.  But  the  water  soon  dries  up  and  the  air 
also  does  not  come  to  it  freely.  But  all  these  objections  are 
obviated  by  the  use  of  the  following  little  apparatus  which  is  at 
once  a  ventilated  moist-chamber  and  a  hanging  drop.  It  was 
Strusburger's53  contrivance. 

Cut  from  common  tough  pasteboard  pieces  like  those  repre- 
sented in  Fig.  110.  The  hole  in  the  centre  should  be  a  little 
smaller  than  the  cover-glass  to  be  used.  Then  put  the  paper, 
pieces  in  water  and  let  them  soak  till  they  are  thoroughly  satur- 
ated. Take  them  out  and  lay  two  or  three  deep  on  a  slide,  and  lay 
the  cover-glass  with  its  hanging  drop  over  the  central  opening. 
The  apparatus  is  now  complete.  I  have  kept  a  hanging  drop 
unchanged  upon  such  an  apparatus  for  a  fortnight  by  simply 
putting  a  few  drops  of  water  from  the  wash  bottle  upon  the  side 
of  the  paper  every  evening.  By  means  of  this  moist-chamber 
I  have  been  easily  able  in  the  spring  of  the  year  to  observe54 
the  Sp'jvogyra  form  its  spores,  while  with  other  apparatus 
I^have  in  most  cases  failed.  With  this  apparatus  too  I  have  had 
much  success  in  cultivating  the  pollen  tubes  in  a  30  per  cent 
solution  of  sugar,  honey  and  like  fluids.55 

Geissler  has  constructed  a  very  peculiarly  formed  moist-cham- 
ber for  examining  objects  in  a  vacuum  or  with  an  atmosphere  of 
carbonic  acid  or  oxygen.  It  consists  of  a  glass  tube  which  is 
widened  and  flattened  in  the  middle  so  as  to  form  a  smooth  disk- 
shaped  space,  the  upper  and  under  walls  of  which  are  brought 


53  Strasburger,  Befruchtnng  n.  Zelltheilung.    Jena,  1878,  p.  5. 

54  See  also  Strasburger.  L  c.,  p.  5,ff. 

53  See  also  Strasbuvger,  1.  c.,  p.  15-25,  especially  p.  1(5.  Here  it  is  said  of  the  culture  of 
the  pollen  of  Pinus  pumilio,  "But  by  the  prevalence  of  bacteria,  of  yeast  cells  and  mould 
fungus,  the  culture  will  be  ruined  at  farthest  in  8  or  10  days.  I  kept  it  the  longest  when  I 
used  thyme  oil  in  a  thousand-fold  dilution  with  10  to  30  per  cent  sugar  solution.  This  ad- 
dition, however,  at  first  hindered  the  formation  of  the  tubes;  but  after  about  two  days 
when  a  part  of  the  thyme  oil  had  evapoi'ated  they  again  began  to  develop  (salicylic  acid  in 
a  1000  fold  dilution  kills  the  pollen  grains),  while  the  increase  of  the  lower  organizations 
which  were  introduced  at  the  same  time  with  the  pollen  grains  was  delayed  for  several 
days. 


252  THE  MICROSCOPE  IN  BOTANY. 

close  together  and  are  of  the  thickness  of  common  cover-glass. 
The  culture  drop  is  brought  between  them.  The  tube  allows  a 
current  of  air  to  be  thrown  around  it  of  any  desired  kind.  This 
apparatus  is  especially  applicable  to  the  cultivation  of  fungus 
spores  and  the  like.53  A  modification  of  the  Greissler  moist- 
chamber  has  recently  been  devised  by  Brefeld.57 

[The  Gas  Slide.  For  the  exposure,  under  the  microscope, 
of  organisms  either  animal  or  vegetable  to  the  effect  of  certain 
reagents  in  the  form  of  gases,  for  the  sake  of  observing  the 
effect  of  these  gases  upon  the  actions  of  living  beings  or  upon 
the  character  of  their  dead  tissues,  it  is  only  necessary  to  have 
a  cell  of  glass,  lying  upon  the  stage,  and  supplied  with  tubes 
for  the  entrance  and  escape  of  the  reagent.  When  constructed 
of  glass  cemented  together,  these  instruments  are  liable  to  sep- 


FIG.  111. 

arate  at  the  joints  and  are  otherwise  especially  subject  to  acci- 
dental injury.  The  form  shown  in  Fig.  Ill,  is  made  almost 
wholly  of  brass,  lies  heavily  and  firmly  upon  the  stage,  and  is 
safe  from  any  considerable  injury  by  breakage.  The  cover- 
glasses  are  easily  accessible  for  cleaning,  and  if  broken  are  easily 
replaced.  Being  metallic  it  transmits  heat  promptly,  in  case  of 
use  upon  the  hot  stage.  It  it  is  made  by  T.  H.  McAllister  of 
New  York,  at  the  suggestion  of  Dr.  T.  H.  Hunt  of  Brooklyn. 
It  consists  of  a  heavy,  slide-shaped  brass  box  with  a  central, 
cylindrical  perforation  20  mm.  wide  and  7  deep.  This  central 
well  is  closed  at  the  bottom  by  a  cover-glass  cemented  to  the 
brass  ledge  on  which  it  rests,  and  is  covered,  after  the  insertion 
and  arrangement  of  the  object,  by  another  cover-glass  which  is 

56Nageli  und  Schwendener,  Das  Mikroskop,  p.  275. 

67  Oscai;  Brefeld,  Culture  method  for  the  investigation  of  fungi.  (In  Botanische  Un- 
tersuchungen  iiber  Schimmelpilze.  Untersuchungeii  aus  dem  Gesammtgebiete  der  My- 
kologie.  Heft  IV,  1881,  p.  1-35).—  See  also  Hansen's  Chambre  lumiide  pour  la  culture  di  a 
organismes  microscopiques.  Avec  deux  figures  dans  le  texte  [Meddelelser  fra  Carlsberg 
Laboratoriet,  p.  18i-183,  Kjobenhavn,  1881.] 


A  LABORATORY  TABLE. 


253 


FIG.  112. 


held  in  place  and  rendered  air-tight  by  a  small  quantity  of  par- 
affine,  oil,  glycerine,  or  other  available  material  around  the  edge. 
Short  brass  tubes  are  provided,  at  the  ends  of  the  apparatus, 
to  be  attached  to  the  tube  bringing  the  gas  to  one  side  of  the 
box  and  conveying  it  away  after  having  passed  through  to  the 
other.  R.  H.  W.] 

[A  Growing  Slide,  or  moist-chamber  and  hanging  drop,  shown 
in  Fig.  112,  is  sold  and  used  in  this  country.  It  consists  of  two 
common  slides  held  together 
with  rubber  bands,  the  upper 
one  perforated  with  a  circular 
hole  10  or  12  mm.  in  diam- 
eter, over  which  the  cover- 
glass  with  the  culture  drop 
is  laid,  being  held  by  a  little 
grease  rubbed  on  about  the  edge  of  the  hole.  When  the 
slide  is  not  under  observation  it  is  laid  in  a  flat  dish  containing 
a  sufficient  depth  of  water  to  overflow  the  lower  slide  and  run 
in  by  capillary  attraction  between  the  two  and  so  prevent  the 
evaporation  of  the  drop.] 

[A  Laboratory  Table.  Prof. 
C.  E.  Bessey,  professor  of  Bot- 
any and  Horticulture  in  the 
University  of  Nebraska,  kindly 
furnishes  me  with  a  plan  of  the 
tables  used  in  the  microscopical 
laboratory  of  that  institution. 
It  is  represented  in  Fig.  113 ;  w 
is  the  window,  a  b  c  e  represents 
the  form  of  the  table,  the  breadth 
of  which  at  a  b  is  1.5  m.,  and  at 
c  e  .6  m. ,  the  perpendicular  length 
1.8  m.  At  the  points  indicated 
FIG  113>  by  x  are  placed  the  micros- 

copes, each  in  such  a  position  as 

to  receive  the  unobstructed  light  from  the  window,  without  lia- 
bility of  interference  from  those  working  at  the  other  micros- 
copes. A.  B.  H.] 


254  THE  MICROSCOPE  IN  BOTANY. 


XIII.     DRAWING  MICROSCOPIC  OBJECTS. 

In  the  introduction  of  this  work  we  have  already  shown  how 
important  it  is  to  permanently  fix  the  microscopic  image  by 
drawing.  Farther  along  we  became  acquainted  with  some  appa- 
ratus by  which  the  image  is  reflected  upon  a  piece  of  paper 
lying  near  the  microscope  so  that  the  drawing  could  be  done  by 
simply  tracing  the  outlines. 

1.    AIDS  TO  MICROSCOPICAL  DRAWINQ. 

An  experienced  draughtsman  can  draw  free-hand  the  sim- 
ple microscopic  images  by  looking  in  the  microscope  now  and 
then,  drawing  a  part  of  the  image  which  he  has  especially  ob- 
served, comparing  the  drawing  with  the  original  to  see  if  it  fully 
corresponds,  then  taking  up  the  next  contiguous  part  and  so  on. 
This  kind  of  drawing  has  this  unqualified  advantage,  that  in 
its  use  one  is  compelled  to  observe  the  image  very  exactly  in 
respect  to  its  forms  and  their  respective  relations.  The  prac- 
tised draughtsman  may  by  this  method  give  to  his  drawing  the 
greatest  perfection.  But  this  chapter  is  written  less  for  the 
skilled  artist  than  for  those  who  are  but  little  if  at  all  proficient 
in  the  pictorial  art.  We  shall  therefore  describe  those  methods 
which  will  assist  the  latter  in  making  graphical  representations. 

To  the  unskilled  it  is  a  matter  of  the  greatest  difficulty  to 
bring  into  proportion  the  large  dimensions  of  the  field  of  view 
with  the  smallness  of  the  object,  and,  further,  to  rightly  estimate 
the  distance  apart  of  the  details  of  the  object  and  to  fix  them. 
These  difficulties  are  overcome  by  the  following  means. 

(a)  Small  objects,  which  are  considerably  extended  in  one 
direction  like  diatoms  and  other  algae,  may  be  drawn  very 
easily  with  the  help  of  the  common  ocular  micrometer.  This 
method  enables  us  to  draw  the  object  in  exactly  the  size  in  which 
it  appears  under  a  certain  definite  magnification.  An  example 
will  immediately  make  this  method  clear. 

I  have  a  diatom,  Pinnularia  viridis  Rabenh.,  to  draw  magni- 
fied 600  times,  Fig.  114.  I  know  that  my  micrometer  scale 


AIDS  TO  MICROSCOPICAL  DRAWING. 


255 


contains  4  millimetres  each  divided  into  20  parts,  and  that  in 
relation  to  my  magnification  of  600  this  length  is  equal  to  0.1 
mm.  (i.e.,  0.1  mm.  in  the  object  covers  the  whole  scale  in  the 
ocular)  ;  the  scale  of  the  micrometer  must  also  with  the  600 
fold  magnification  appear  to  be  60  mm.  long.58  I  draw  the  scale 
in  this  length  on  a  piece  of  paper,  Fig.  114,  dividing  the  whole 
scale  into  8  equal  parts,  each  =  7.5  mm.,  and  these  in  halves. 
The  long  marks  0,  10,  20,  etc.,  correspond  to  every  ten  divisions 
of  the  micrometer,. the  lines  between  standing  for  five  divisions. 
I  have  already  ascertained  that  the  diatom  measures  0.137  mm. 
which  by  600  fold  magnification  must  give  a  length  of  82  mm.  ; 
by  that  I  can  easily  fix  the  position  of  the  points  e  and  f,  with 
the  millimeter  scale.  I  now  bring  the  microscopic  image,  and 


70     80 


FIG.  114. 

the  scale  in  the  ocular  of  the  microscope  into  such  a  position  as 
is  shown  in  the  illustration,  Fig.  114.  Now  I  can  easily  fix  the 
outline  of  the  diatom  and  the  position  of  its  separate  parts  on 
the  drawing  of  my  scale  with  the  greatest  exactness.  Three 
markings  of  the  Pinnularia,  for  example,  correspond  with  one  of 
the  divisions  of  5  on  my  scale.  The  two  halves  of  the  inter- 
rupted middle  line  are  3  divisions  of  the  scale  apart  at  the  center. 
The^  various  dimensions  of  the  picture  will  be  made  to  corres- 
pond to  the  reality  since  they  are  all  laid  out  like  a  chart  on  a 
network  of  fixed  lines.  This  kind  of  drawing  is  preferable  to 
that  with  the  camera  lucida  for  diatoms  of  very  delicate  frustules 
since  the  delicate  markings  of  these  forms  are  seen  with  great 
difficulty  in  the  reflected  image. 

68  This  value  will  naturally  differ  with  every  microscope  aud  for  each  maguiflcatiou. 


256  THE  MICROSCOPE  IN  BOTANY. 

(b)  111  the  drawing  of  those  images  that  extend  uniformly 
over  the  field  of  view,  the  aid  of  the  micrometer  scale  is  not 
nearly  as  suitable.  In  this  case  drawing  by  free-hand  is  fa- 
cilitated by  the  use  of  an  ocular  in  whose  diaphragm  are  cross 
threads  which  divide  the  field  into  four  parts.  One  with  double 
cross  threads  like  the  form  shown  in  Fig.  115  is  still  better. 
Since  this  can  seldom  be  bought  I  will  explain  how  it  can  be 
made.  Prepare  a  circular  rim  of  brass  about  the  size  of  Fig. 
115,  which  may  be  fitted  to  the  ocular  tube  from  above.  On  this 
the  cross  threads  may  be  made  fast,  which  in 
low  power  oculars  may  be  made  of  fine  glass 
threads  drawn  out  by  means  of  the  blow  pipe 
flame,  or  of  human  hair  previously  boiled  in 
alcohol  to  get  the  oil  out.  In  high  power  oculars 
the  cross  threads  should  be  made  of  spider's 
web.  The  preparation  of  the  latter  is  by  no 
means  so  difficult  as  is  generally  supposed,  only  one  must  not 
stretch  them  —  and  the  same  is  true  of  hairs  —  when  the  air  is 
dry,  since  afterwards  the  fiber  by  taking  up  moisture  in  a  humid 
atmosphere  would  contract  and  break.  First  mark  the  places 
on  the  brass  rim  where  the  fibers  are  to  go  and  then  fasten  one 
end  on  one  side  by  means  of  wax  and  draw  the  thread  over  to  the 
other  side,  fastening  it  in  the  same  way,  warming  the  wax  to 
make  it  soft.  Having  made  the  fibers  fast  on  the  rim  put  some 
Canada  balsam  on  the  lower  side  of  it  and  drop  it  carefully  into 
the  ocular  down  iipon  the  diaphragm  where  it  will  stick  fast. 
After  the  Canada  balsam  is  dry  screw  on  the  eye  lens  and  after- 
wards remove  it  as  seldom  as  possible. 

How  then  shall  we  apply  this  contrivance  to  the  drawing  of 
microscopical  pictures?  Fig.  116  represents  a  schlerenchyma 
bundle  in  a  cross  section  of  the  root  of  Pteris  aquilina  which 
we  will  draw  by  means  of  the  double  cross  threads  magnified 
600  diameters.  This  is  easily  done  when  we  have  first  learned 
the  size  of  the  square  by  direct  measurement.59  In  the  other 
drawing  with  this  magnification,  the  length  of  our  micrometer 

69  Lay  the  crossed  threads  under  the  microscope  as  an  object  screw  on  a  low  power  n, 
and  draw  it  in  its  natural  size  with  a  camera  lucida.  The  length  of  the  side  is  then  meas- 
ured with  the  millimeter  scale  and  divided  by  n;  the  quotient  gives  the  exact  length  in 
millimeters. 


AIDS  TO  MICROSCOPICAL  DRAWING. 


257 


scale,  4  mm.  as  it  appeared  in  the  ocular  was  equal  to  0.1  mm. 
used  as  an  object,  and  magnified  600  times.  We  have  also  ascer- 
tained by  measurement  that  the  length  of  one  side  of  the  mid- 
dle square  is  2.2  mm.  Consequently  under  the  same  conditions 
this  side  will  appear  to  be  33.  mm.  long,  for : 
4:0.1  X  600:  :2.2:z 

cc  =  33. 

Now  we  remark  that  the  upper  side  of  the  square  «?,  Fig.  116, 
covers  three  cells.     It  will  be  easy  to  estimate  their  respective 


FIG.  116. 

lengths  and  to  fix  the  points  5,  c,  d,  e,f,  g,  also  like  points  on  the 
other  three  squares,  and  the  uniting  points  of  the  cellular  network 
which  fall  inside  the  squares  i,  7^,  k,  rr^  are  likewise  determined 
without  difficulty  by  comparative  estimation.  When  they  are 
all  determined  for  a  given  cell  then  the  outline  will  be  drawn, 
and  so  on.  In  this  way  we  are  able,  without  very  great  errors, 
to  map  out  a  group  of  cells  like  these,  and,  what  is  of  greater 
importance,  we  soon  attain  by  this  method  a  certain  readiness 
and  skill  in  estimating  sizes  and  distances  under  the  microscope. 

17 


258  THE  MICROSCOPE  IN  BOTANY. 

This  readiness  will  very  often  be  of  use  to  the  microscopist. 
On  the  right  side  of  the  illustration,  the  network  of  cells  is 
shown  drawn  in  outline ;  the  rest  of  the  cells  are  more  fully 
drawn. 

(c)  In  most  cases  when  a  microscopic  image  is  to  be 
traced,  recourse  will  be  had  to  some  camera  lucida  already 
described.  The  use  of  this  very  helpful  apparatus  is  very  soon 
and  very  easily  learned.  There  is  therefore  need  of  saying  but 
a  few  words  about  it. 

The  eye  should  be  held  close  to  the  opening,  provided  for 
seeing  in  the  instrument  and  look  perpendicularly  down,  for  by 
looking  obliquely  the  image  may  be  considerably  distorted. 
The  paper  upon  which  the  drawing  is  to  be  made  should  be 
fastened  lying  flat,  at  a  standard  distance  of  25.4  cm.  from  the 
camera  lucida.  It  is  best  to  provide  a  drawing  board  on  which 
the  paper  may  be  fastened  and  which  may  be  placed  at  this  dis- 
tance from  the  camera. 

In  order  to  draw  a  picture  by  means  of  the  camera  lucida  with- 
out painfully  straining  the  eyes,  it  is  necessary  that  the 
microscopic  image,  and  the  paper  and  pencil  be  uniformly 
illuminated.  If  the  image  has,  in  comparison  with  the  paper, 
too  strong  a  light  the  pencil  will  be  seen  with  difficulty  if  at  all. 
On  the  contrary,  if  the  paper  in  comparison  to  the  image  be  too 
strongly  illuminated,  the  delicate  outlines  of  the  latter  will  be 
indistinct.  The  first  usually  occurs  where  the  image  of  the 
paper  and  pencil  is  thrown  into  the  field  of  view  of  the  micro- 
scope and  the  latter  when  the  microscopic  image  is  reflected 
upon  the  paper.  This  difficulty  may  be  remedied  by  throwing 
either  the  image  or  the  paper  into  a  shadow.  Both  may  be  done 
simply  with  the  hand,  or  by  a  properly  constructed  screen  of 
paper,*  or  by  a  disk  of  pasteboard  set  up  at  some  distance,  and 
the  like.  Hartnack  provides  his  cameras  with  a  blue  glass  plate, 
which  partly  obscures  the  light,  and  this  appears  in  fact  to  have 
been  applied  to  the  construction  of  many  cameras.60  A  few 
trials  with  the  microscope  with  different  magnifications  will 
afford  the  necessary  experience  for  properly  managing  the  light. 

*  See  Note,  page  116,  for  description  of  a  handy  form. 

6°  See  C.  Cramer  in  Botan.  Centralbl.    1881,  Bd.  VII,  pp.  3SXJ91. 


CONDUCTING  MICROSCOPICAL  DRAWING.  259 

Iu  tracing  the  outlines  of  the  image  under  the  camera,  the 
pencil  used  should  not  be  too  hard  and  the  lines  should  be  very 
light,  and  then  they  will  often  appear  rough,  for  the  position 
which  one  has  to  take  in  this  work  is  not  very  favorable  to  nice 
drawing. 

2.     CONDUCTING  MICROSCOPICAL  DRAWING. 

How  a  microscopical  drawing  should  be  carried  out  depends 
upon  what  relations,  qualities  and  observations  we  wish  pictori- 
al ly  to  express.  We  have  already  remarked  that  a  microscopical 
anatomical  drawing  should  by  no  means  be  a  mere  copy  of  the 
image  seen,  but  that  it  should  reproduce  the  sum  of  the  experi- 
ences which  the  observer  has  had  with  his  preparation.  Fur- 
thermore, in  most  cases  the  drawing  should  show  only  those 
relationships  which  the  observer  has  arrived  at  by  his  analysis ; 


FIG.  117. 

hence  it  will  often  not  by  far  show  all  of  those  parts  which  are 
in  reality  seen  in  the  microscopic  image.  Suppose,  for  example, 
that  one  makes  a  purely  histological  study  in  order  to  represent 
the  relative  position  of  the  cells  in  the  tissue ;  certainly  he  will 
not  need  to  draw  the  contents  of  each  single  cell,  its  protoplasm, 
nucleus,  etc.,  or  the  finer  markings  of  the  cell-wall  which  may 
temporarily  interest  him.  Take  a  concrete  example.  Some 
one  studies  the  anatomy  of  the  stem  of  Richardia  africana. 
He  wishes  to  know  the  vascular  bundles  in  respect  to  their  form, 


260 


THE  MICHOSCOPE  IN  BOTANY. 


their  position  and  their  structure.  In  this  case  it  will  be  nec- 
essary to  draw  only  the  outlines  of  the  cells,  and  their  respective 
attachments  and  perhaps  also  the  relative  thickness  of  their  walls. 
His  drawing,  Fig.  117,  will  therefore  represent  the  cell  walls 
with  single  lines  not  doubly  outlined  as  they  are  in  fact.  He 
will,  perhaps,  in  order  to  make  it  perfectly  understandable  to 
another,  express  the  cell  walls  of  the  surrounding  parenchyma 
.tissue  by  the  more  delicate  lines,  and  the  cambiform,  by  the 
stronger  lines,  and  the  vascular  walls  by  the  strongest,  or  by 
double  outlining.  Such  a  drawing  is  naturally  in  the  high- 
est decree  diagrammatic,  but  it  perfectly  satisfies  all  demands 


FIG.  us. 


made  upon  it,  and  is  to  be  preferred  to  any  drawing  which  with 
a  mere  photographic  fidelity  reproduces  the  microscopic  image, 
in  that  it  does  not  divert  the  eye  from  the  principal  thing  by  the 
presence  of  unessential  accessories. 

Take  another  example  by  which  we  can  see,  in  the  same  illus- 
tration, the  distinction  between  a  diagrammatic  and  a  completed 
drawing.  Fig.  118  represents  a  highly  magnified  trans-section 
through  the  upper  part  of  the  stomata,  and  the  adjacent  epi- 
dermis, of  the  needle  of  Taxus  baccata.  The  left  half  of  the 
drawing  is  diagrammatic,  the  other  is  completed.  The  left  teaches 
us  to  know  only  the  form  and  size  of  the  cells,  the  substance  of 
the  cell  wall  and  the  casual  parting  of  the  protoplasmic  cell 
contents.  The  right  half  shows  us  all  these  relations,  and  further 


CONDUCTING  MICROSCOPICAL  DRAWING. 


261 


the  extension  and  form  of  the  cuticle,  the  structure  of  the  cell 
wall,  the  intercellular  substance,  and  the  different  appearances  of 
the  cell  contents  in  the  stomata-closing  cells,  the  epidermis  and 
the  sub-epidermal  layer.  The  right  half  shows  us  all  the  relations 
which  we  are  able  to  deduce  from  the  portion  of  the  preparation 
examined  and  with  the  magnification  employed. 

It  is  self-evident  that  one  may  make  a  microscopical  drawing 
more  or  less  diagrammatic  according  to  the  requirements  of  each 
case.  Fig.  119  represents  the  wood  cells  of  a  young  coniferous 
plant.  I  is  the  most,  III  the  least,  diagrammatic.  In  I  the  middle 


III 


lamella  is  represented  by  a  simple  thin  line,  while  the  inward 
thickening  layer  is  indicated  by  a  heavy  single  line  ;  the  wood 
layer  between  is  not  designated  further.  In  II  the  middle  lamella 
is  indicated  by  two  delicate  lines,  the  inner  layer  by  a  delicate  and 
a  heavy  line,  the  latter  being  the  boundary  line  of  the  cell  cavity 
while  the  woody  layer  is  not  further  exhibited.  In  III  at  last 
the  concentric  layers  of  the  latter  are  represented.  A  quite 
perfect  picture  of  the  cell  wall  structure  is  shown  in  this  drawing, 
as  one  sees  it  with  a  hteh  magnification. 

O  O 

The  representation  of  cell  walls  in  microscopical  drawing  is 
by  no  means  difficult  since  all  that  is  required  is  uniform  con- 
centric outlines.  The  difficulty  of  representation  increases  when 
fluid,  or  semifluid  cell  contents  are  to  be  pictured.  If  the  cell 
contents  are  a  clear,  homogeneous  fluid, we  must  depart  from  a 


262 


THE  MICROSCOPE  IN  BOTANY. 


strictly  pictorial  representation  since  a  fluid  shows  itself  under 
the  microscope  only  by  its  refraction  of  light.  At  most  it  can 
be  (Ungrammatically  represented  only  by  shading  or  laying  in 
with  the  rubber.  But  it  is  otherwise  with  the  protoplasm  and  its 
granules  and  air  bubbles,  which  may  be  very  well  and  very 
beautifully  reproduced  by  drawing.  It  may  be  done  either  with 
the  drawing  pen  and  India  ink  or  with  the  lead  pencil.  Suppose 
we  are  to  draw  the  protoplasmic  contents  of  one  of  the  upper, 
right  hand  cells  of  Fig.  118.  We  first  make  the  outlines  of  the 
granular  substance  with  fine  points.  Then  we  fill  in  the  space 
pretty  uniformly  with  very  delicate  points.  Then  in  those 
places  which  in  the  microscope  appear  to  be  more  densely  gran- 
ulated we  add  a  corresponding  quantity  of  points,  so  that  they 
stand  thicker  and  here  and  there  touch  each  other.  And  finally 
the  larger  and  largest  grains  are  represented  by  minute  circles 
or  irregular  outlines  according  to  the  appearance  of  the  object. 
If  the  protoplasm  is  very  thick  and  cloudy  it  will  not  do  to  make 
the  background  of  delicate  little  points,  but  it  should  be  made 
of  many  finely  entangled  lines  running  through  and  through 
each  other  as  is  seen  in  the  lower  cells  on  the  right  hand  side  of 
Fig.  118. 

In  the  examples  thus  far  treated  we  have  dealt  with  the  re- 
production of  images  which  are  seen  with  a  single  adjustment 
of  the  microscope,  but  we  shall  often  be  required 
to  combine  several  adjustments,  and  so  make  a 
drawing  which  at  least  in  places  represents  corpo- 
real thickness.  As  is  well  known,  this  can  be 
accomplished  only  by  the  addition  of  shading,  and 
nowhere  will  there  be  more  faults  committed  than 
right  here,  as  Nageli  has  well  said.  In  botanical 
literature  there  are  many  drawings  of  this  kind, 
in  which  the  body  is  represented  as  at  the  same 
time  transparent  and  untransparent,  as  flat  and 
wavy,  and  as  round  and  angular.  In  order  to 
draw,  at  least  npproximately  right,  we  must  think 

always  of  a  definite  direction  from  which  the  li<rht 
FIG.  120. 

falls  upon   the  body  to  be  shaded,  for  example, 

from  the  right  or  left  above  at  an  angle  of  45°. 


CONDUCTING  MICROSCOPICAL  DRAWING. 


263 


Yeiy  often  partly  corporeal  representations  are  of  spiral  tissue 
such  as  is  pictured  in  Fig.  120.  We  immediately  see  from 
the  drawing  that  the  wall  of  the  tube  is  not  here  corporeally  or 
perspectively  represented,  but  only  the  inclosed  spiral  band. 
The  walls  are  given  as  they  would  be  seen  by  one  medium  ad- 
justment of  the  lens,  but  the  spiral  band  represents  several 
adjustments  combined  into  one  view.  It  supposes  the  light  to 
enter  from  the  upper  right  hand  side. 


FIG.  121. 

Shading  is  particularly  required  where  it  is  desired  to  represent 
any  cell-contents  which  have  a  peculiar  form.  Included  in  this  are 
chiefly  grains  of  starch,  chlorophyll  and  coloring  matter,  likewise 
crystals  and  drops  of  fluid  and  air-bubbles  in  fluids.  Little  need 
be  said  concerning  the  representation  of  starch  grains  since  they 
occur  in  many  forms  simple  and  compound.  As  an  illustration 
we  give  a  picture  of  the  well-known 
starch  grain  of  the  potato,  Fig.  121 ; 
the  illustration  shows  two  single  grains 
of  starch  which  are  shaded  as  if  the 
light  fell  from  above  in  an  oblique  di- 
rection. It  is  impossible  by  this  kind 
of  shading  to  make  the  impression 
that  the  grain  is  a  flat  disk,  as  by  a 
want  of  shadow  it  might  easily  appear 
to  be.  Chlorophyll  grains  must  be 
represented  commonly  as  rough  little 
balls,  as  shown  in  Fig.  122,  I.  If  we  wish  to  show  highly 
magnified  chlorophyll  grains  with  starch  grains  on  the  inside, 


FIG.  122. 


264 


THE  MICHOSCOPE  IN  BOTANY. 


we  have  recourse  to  the  forms  indicated  in  the  lower  granules 

O 

of  Fig.  122,  II.  Small  solid  granules  of  another  kind  occur 
more  rarely,  for  example  in  the  yellow-colored  floral  leaves. 
If  very  small  they  are  represented  by  simple  circles,  as  in 
Fig.  123,  which  is  a  longitudinal  section  of  the  head  of  the 

nectar  hair  of  Parnassia  pdlustris. 
If  they  are  larger  they  may  have 
the  proper  shading  of  a  globe  as 
indicated  in  the  sketch,  Fig.  123. 
Crystals  which  occur  in  plant  cells, 
must  always  be  drawn  solid,  in 
order  to  make  the  right  impression 
upon  the  beholder ;  they  may  be 
shown  in  outline,  or,  what  is  better, 
shaded.  Of  the  manifold  forms 
of  microscopic  crystals,  we  give  in 
Fig.  124  the  three  which  occur 
most  frequently,  in  order  to  exhibit 
the  kind  of  shading  to  be  used. 
It  becomes  clear  from  the  shading 
that  the  middle  figure  in  the  illus- 
tration is  a  quadra-octahedral  crystal  with  truncated  point ;  the 
one  on  the  right,  on  the  contrary,  is  a  rhomboid. 

The  representation  of  small  drops  of  fluid  or  of  air  bubbles  is 
attended  with  more  difficulty  since  their  appearance  changes 
with  the  adjustment  of  the  objective.  Focussed  in  the  middle 
an  air  bubble  appears  sharp  and 
with  a  double  outline,  while  if 
the  focussing  is  higher  up  it  as- 
sumes the  form  of  a  dark  colored 
globe  which  becomes  brighter 
toward  the  poles,  and  at  the  pole 
itself  brightly  sparkles.  The  pictorial  representation  is  common- 
ly by  two  concentric  circles  which  stand  apart  about  as  the  out- 
lines are  perceived  to  do  with  the  medium  adjustment  of  the  lens. 
It  need  scarcely  be  added  that  the  foregoing  analysis  includes 
only  a  few  of  the  more  frequently  occurring  cases.  On  the 


FIG.  123. 


FIG.  124. 


DRAWING  MATERIALS.  265 

other  hand,  we  must  pass  over  to  the  practical  draughtsman  a 
great  many  things,  to  find  out  for  himself  the  best  way  of  drawing 
them.  He  will  be  much  helped  by  carefully  studying  good 
drawings  of  objects  which  are  like  his  preparations.  Of  works 
which  contain  the  best  illustrations  for  this  purpose  we  commend 
Sachs'  Text  Book  of  Botany ;  DeBary,  in  Sachs',  Hofmeister's, 
and  DeBary's  Manuals  ;  Ltirssen's  Medico-pharmaceutical  Bot- 
any ;  Strasburger's  "Cell-formation  and  Cell-division";  Kny's 
Botanical  wall  charts  ;  like  wise  numerous  illustrated  monographs 
in  Priugsheim's  Year  Book  and  in  the  "Botanischen  Zeitung." 


3.    DBA  WING  MATEKTAT.S. 

Most  anatomical  drawing  will  be  done  with  the  lead  pencil 
which  should  not  be  too  hard  if  one  wants  delicate  soft  pictures. 
Faber's  Xos.  2  and  3,  are  best  and  especially  the  yellow  hexa- 
gonal forms.  The  pencil  should  have  a  very  fine  point  and 
this  can  be  best  made  by  the  use  of  a  file.  Shadings,  cloudy 
parts,  delicate  cell-contents  can  be  most  conveniently  put  iu 
by  means  of  a  soft  leather  rubber,  which  gives  to  the  drawing 
a  very  neat  and  at  the  same  time  natural  appearance. 

For  drawings  that  are  to  serve  as  guides  in  lithographing  it 
is  better  to  use  the  drawing-pen  and  India-ink,  rather  than  the 
pencil,  thus  making  the  so-called  "stipple  drawing  "  so  like  the 
lithograph  that  one  may  be  reasonably  assured  that  no  strange 
additions  will  be  made  to  it  iu  lithographing  it.  India-ink 
drawings  also  serve  well  for  photographic  transfers,  but  for  this 
purpose  cinnabar  color  should  be  mixed  with  the  India-ink. 

For  coloring  microscopical  drawings  either  oil-crayons  or 
water  colors  are  most  to  be  commended.  In  the  use  of  the  oil- 
crayons  the  paper  does  not  need  to  be  previously  touched  up 
with  the  rubber  eraser.  Sussuers'  oil-crayons  allow  different 
colors  to  be  drawn  over  each  other  so  that  one  can  obtain  with 
them  any  desired  shade.  In  respect  to  water  colors,  the  fluid 
colors  in  tubes  are  to  be  preferred  before  all  others.  Iu  recent 
times  they  have  been  extensively  applied  to  other  purposes. 


266  THE  MICROSCOPE  IN  BOTANY. 

. 

For  putting  on  colors  use  the  sable  pencil,  or  the  common  India- 
ink  pencil  with  a  long  handle. 

Of  the  kind  of  paper  to  be  used  choose  a  good  number  of 
"Watman"  which  is  not  too  rough.  For  India-ink  drawing  a 
well  smoothed  cardboard  is  best.  For  preliminary  sketches 
make  a  good  quality  of  writing  paper  do,  as  the  Watraan  paper 
is  too  expensive. 


MICROSCOPICAL  EEAGENTS.  267 


CHAPTER  IV. 

MICROSCOPICAL  REAGENTS. 

I.     INTRODUCTION. 

IF  we  make  a  section  through  some  plant  tissue,  for  example, 
a  cross  section  through  a  young  wood  stem,  and  examine  it  under 
the  microscope  in  water,  glycerine,  or  other  uncolored  fluid  it 
will  —  with  the  exception  of  a  few  unimportant  cases  —  appear 
throughout  uncolored.  This  coloiiessness  will  show  itself  not 
only  in  the  membranes  and  walls  of  most  cells,  but  also  in  most 
of  the  fluid  and  solid  contents  of  the  cells.  It  is  obvious  that 
it  would  frequently  be  very  difficult  to  determine,  at  sight,  as  to 
the  nature  of  the  small  bubbles,  drops  and  granules  in  the  in- 
terior of  the  cells.  It  is  quite  otherwise  with,  for  example,  the 
widely  distributed  chlorophyll  which  by  reason  of  its  green  color 
is  very  easily  recognized,  even  by  the  beginner.  If,  on  the  other 
hand,  the  small  drops  of  oil  and  fat,  the  granular  substances 
containing  proteids,  etc.,  were  provided  with  a  color  peculiar  to 
themselves,  they  would  be  much  more  easily  identified  than  they 
now  are. 

Quite  early,  indeed  in  the  first  years  of  our  century,  it  was 
observed  that  if  a  fluid  containing  free  iodine  in  solution  were 
added  to  cells  containing  starch  grains,  the  grains  became 
blue  under  the  influence  of  the  iodine  while  the  other  parts  of  the 
cell-body  remained  uncolored,  or  were  at  least  of  another  color 
than  blue.  Nageli  afterwards  showed  that  the  blue  coloring  was 
produced  by  the  particles  of  iodine  entering  the  starch  grain 
with  a  characteristic  molecular  intercalation. 

Soon  after  that  Theodor  Hartig  found  that  an  ammoniacal 
solution  of  carmine  behaved  in  a  very  remarkable  way  towards 
the  protoplasmic  contents  of  the  cell,  also  to  the  nucleus  and  to 
the  protoplasm  in  the  restricted  sense  of  the  term.  "While  these 


268  THE  MICROSCOPE  IN  BOTANY. 

substances  in  a  living  state  behaved  themselves  quite  indifferently 
towards  the  red  colored  fluid,  the  nucleus  immediately  after  death 
eagerly  absorbed  the  carmine  substance,  and  in  consequence  was 
stained  a  beautiful  carmine  red.  While  previously  the  nucleus 
of  many  cells  could  be  recognized  with  the  greatest  difficulty, 
they  were  now  observed  without  trouble.  The  dead  protoplasm 
at  first  continued  perfectly  colorless  under  the  influence  of  the 
carmine  fluid,  but  after  a  long  time  it  absorbed  the  carmine  color, 
yet  with  a  much  more  delicate  shade  than  the  nucleus. 

It  has  long  been  known  to  the  chemist  that  ferrous  salts  which 
contain  but  very  small  traces  of  ferric  oxide,  enter  into 
complete  chemical  combinations  with  tannic  acid,  which  are  partly 
distinguished  in  aqueous  solutions  in  the  form  of  dark  blue  or 
dark  green  precipitates.  So,  when  cells  containing  tannic  acid 
are  impregnated  with  this  iron  compound,  a  precipitate  is 
immediately  produced  which  has  this  color  and  demonstrates 
the  presence  of  tannic  acid. 

From  these  few  examples  it  follows  that  there  are  certain 
substances  available  to  the  microscopist  by  which  he  is  able  to 
give  account  of  the  nature  of  the  compound  materials  of  plants. 
These  substances  are  called  microscopical  reagents,  and  the  effect 
which  they  produce  upon  the  parts  of  the  plant  is  designated  the 
reaction,  and  the  methodical  application  of  the  reagents,  in  order 
to  recognize  the  nature  of  the  framework  or  the  contents  of  the 
cell,  microscopical  analysis. 

The  effects  of  microscopical  reagents  are  of  various  kinds. 
If  the  reagent  unites  with  the  material  to  be  investigated  form- 
ing new  combinations,  the  effect  in  this  case  is  chemical.  The 
reaction  described  just  now  on  tannic  acid  is  founded  on  a 
chemical  effect.  If  proteid  substances  are  treated  with  nitric 
acid  and  ammonia  there  is  produced  with  a  brown  coloring  a 
xanthoproteid  salt  of  the  alkali.  This  is  another  example  of 
the  effect  of  a  chemical  reagent. 

A  great  many  microscopical  reagents,  however,  produce 
effects  upon  the  substances  under  investigation  very  different 
from  those  described  above;  namely,  physical  effects.  For  ex- 
ample, we  lay  a  transverse  section  of  the  young  stem  of  Lonicera 
in  a  mixture  of  aniline  red  (ftichsin)  and  aniline  blue  dissolved 


MICROSCOPICAL  REAGENTS.  269 

in  alcohol  for  a  short  time,  then  pass  it  through  absolute  alcohol 
and  wash  it  out  in  distilled  water  and  put  it  under  the  microscope 
for  examination  in  water  or  glycerine,  it  will  show  that  the 
different  layers  of  fibers  have  absorbed  in  part  different  colors. 
The  walls  of  the  outer  layer  of  bark  have  become  slightly  bluish, 
while  the  inner  bark  layer  remains  almost  colorless.  The  bast 
vessels  and  a  part  of  the  soft  bast  appear  reddish.  The  cam- 
bium zone  is  perfectly  uncolored.  •  The  vascular  bundles  are  a 
beautiful  violet,  the  wood  parenchyma  only  being  uncolored.  The 
outer  layers  of  pith  are  dark  blue,  the  inner  quite  uncolored. 
These  different  colorings  rest  upon  the  fact  that  the  red  and  blue 
stains  have  mechanically  penetrated  those  cell  walls  which  have 
a  certain  structure.  There  has  taken  place  a  molecular  interca- 
lation in  the  tissue  of  the  wralls  but  no  chemical  combination  with 
the  substance  of  them.  The  uncolored  parts  possess  a  certain 
physical  quality  which  does  not  admit  either  of  the  pigments  to 
penetrate  them,  the  red  colored  absorb  the  fuchsin,  the  blue 
only  the  aniline  blue,  while  the  violet  takes  up  both.  It  can  be 
easily  shown  that  no  chemical  change  has  taken  place  in  the  cell 
wall  by  reason  of  the  action  of  the  coloring  substances.  If  the 
section  be  laid  for  a  considerable  time  in  absolute  alcohol  the 
colors  entirely  disappear.  They  have  been  leached  out  by 
the  alcohol.  Now  the  section  will  submit  to  any  other  reagents 
giving  precisely  the  same  reactions  as  though  it  had  not  been 
colored  at  all. 

Since  a  large  number  of  microscopical  reagents  exercise  a 
purely  physical  effect  it  is  essentially  false  to  speak  of  a  micro- 
chemical  analysis  or  to  designate  the  doctrine  of  microscopical 
reagents  and  their  application  with  the  expression  "micro-chem- 
istry." 

Whether  a  microscopical  reagent  produces  a  chemical  or  phys- 
ical effect  is  a  matter  of  no  distinct  consequence,  and  micro- 
scopical analysis  attributes  to  both  categories  of  reagents  the 
same  value.  The  problem  is  first  to  find  ways  and  means  for 
"establishing  the  chemical  constitution  of  the  substances  of  which 
plants  are  made,  and  second  to  make  the  delicate  structural 
relations,  in  the  investigation  of  which  we  come  upon  our  chief 
difficulties,  stand  out  more  sharply.  It  accomplishes  this  mainly 


270  THE  MICROSCOPE  IN  BOTANY. 

in  one  way.  It  is  naturally  self-evident  that  the  microscope 
must,  in  every  single  case  (if  it  be  not  otherwise  possible),  make 
it  clear  what  the  effect  of  the  reagent  which  we  employ  really 
is,  though  it  need  not  be  concealed  that  we  still  know  very  little 
of  the  way  and  manner  of  the  working  of  many  reagents. 

Microscopical  reagents  and  their  effects  constitute  a  purely 
empirical  science.  We  are  indebted  to  experiments  for  the  pos- 
session of  most  of  the  reagents.  Those  discovered  by  scientific 
deduction  are  the  small  minority.  It  is  not  to  be  denied  that  a 
great  many  proposed  reagents  are  of  no  very  great  im- 
portance, and  give  but  doubtful  results.  The  effect  of  others 
which  have  been  much  commended  by  their  discoverers  has 
not  yet  been  sufficiently  verified  to  allow  them  without  further 
trial  to  be  set  down  as  good  reagents.  Microscopical  analysis 
generally  is  of  too  recent  a  date  to  be  considered  in  any  respect 
concluded ;  on  the  contrary,  its  wider  cultivation  belongs  to  the 
future. 

The  older  phytotomists  indeed  employed  single  reagents  now 
and  then,  but  it  was  clone  only  incidentally  and  the  results  were 
considered  of  no  great  importance.  The  first  who  emphasized 
the  eminent  significance  of  a  methodical  application  of  micro- 
scopical reagents  was  Theodor  Hartig.  By  their  aid  he  came 
to  know  many  anatomical  relations  which  escaped  the  notice  of 
his  contemporaries  and  which  were  generally  rejected  by  them, 
for  Hurting  constructed  a  nomenclature  of  his  own  which  dif- 
fered much  from  that  of  other  botanists,  and  he  gruffly  rejected 
all  views  opposed  to  his  own.  Not  until  recent  times  has  it 
come  to  be  recognized  more  and  more  that  Hartig  was  far  in 
advance  of  his  contemporaries  in  the  interpretation  of  many 
anatomical  structural  relations.  He  came  to  a  right  knowledge 
of  these  in  many  cases  by  the  use  of  microscopical  reagents. 
The  number  of  botanists  who  afterwards  essentially  contributed 
to  the  cultivation  of  microscopical  analysis  is  quite  large.  We 
shall  become  acquainted  with  their  individual  contributions  in 
the  course  of  this  and  the  following  chapters.  Here,  first  of  all, 
may  be  given  those  names  which  take  the  most  conspicuous 
places  in  the  history  of  microscopical  analysis.  Nageli  is  first 
to  be  mentioned,  who  By  his  classical  investigations  of  the  in- 


PREPARATION  OF  REAGENTS.  271 

fluence  of  iodine  reagents  upon  starch,  cellulose,  etc.,  produced 
a  model  work ;  then  came  Sachs  who  found  a  series  of  new  and 
beautiful  reactions  and  methodically  applied  the  same  in  extra- 
ordinary investigations  of  the  germination  of  seeds ;  next  was 
Hanstein  to  whom  we  are  indebted  likewise  for  a  number  of  im- 
portant reactions.  Finally,  we  name  Strasburger  and  Wiesner 
whose  numerous  discoveries  in  tjiis  line  will  be  enumerated 
further  on. 

Statements  concerning  the  methods  of  microscopical  reactions 
are  found  widely  distributed  in  anatomical  and  physiological 
literature.  With  the  exception  of  an  early  list  of  reagents 
by  Theodor  Hartig1  a  serviceable  catalogue  of  them  has  never 
been  attempted.  There  are  to  be  sure  in  the  works  of  Nageli 
and  Schwendener,  Dippel,  Frey  and  others,  so  often  cited  above, 
many  incidental  statements  upon  this  subject,  but  they  are  either 
very  short  or  very  hard  to  find,  or  are  no  longer  accepted. 
While  the  preparation  of  these  chapters  was  going  on,  there 
appeared  a  very  careful  brief  compendium  of  the  facts  belonging 
to  this  subject  by  V.  A.  Poulsen  under  the  title  "Botanisk 
Mikokemi,  Yejledning  ved  fytohistologiske  Undersogelser  til 
Brag  for  studerende."2  We  can  commend  this  little  work  in 
the  warmest  terms  for  the  instruction  of  all  beginners  in  that 
department. 

In  the  remaining  part  of  this  treatise  we  propose  to  furnish 
a  complete  survey  ot  microscopical  reagents,  their  preparation, 
application,  and  the  effect  which  they  produce  upon  the  organs 
of  the  plant.  In  the  great  quantity  of  data  which  had  been. 
wrought  out  it  was  not  possible  to  name  all  the  known  facts, 
not  indeed  'because  it  would  thereby  unreasonably  increase 
the  size  of  the  work.  There  is  a  whole  series  of  reactions 
whose  usefulness  is  by  no  means  securely  established  which  must 
be  excluded  for  this  reason.  Attention  can  be  drawn  to  them 
only  by  brief  references  to  the  literature  which  treats  of  them. 
In  the  main,  the  author  has  striven  to  give  all  references  to  the 
related  literature  with  the  utmost  completeness,  in  order  to  spare 

1  Hartig,  Entwicklungsgeschichte  des  Pflanzenkeims.    Leipzig,  1858,  pp.  153-150. 

2  Kopenhagen,  1880. — Also  appeared  in  a  German  translation  by  C.  Miiller,  1881.  Also  an 
English  translation  of  the  German  edition,  1883,  published  by  S.  E.  Cassino  and  Co.,  Boston, 
Mass. 


272' 


THE  MICROSCOPE  IN  BOTANY. 


the  reader  time-consuming  and  often  fruitless  searching.  When- 
ever it  was  possible  the  reactions  were  carefully  tested,  as  will 
be  seen  by  a  number  of  records  relating  thereto. 


II.   APPARATUS  FOR  THE  PREPARATION  OF 
REAGENTS. 

In  almost  all  cases  the  microscopist  must  prepare  his  own 
reagents.  Particularly  is  this  necessary  if  he  desires  them  to 
be  absolutely  pure.  The  reagents  are  simple  fluids,  or  mixtures 
of  several,  or  finally  solutions  of  solid  substances  in  simple 
fluids  or  fluid  mixtures. 

The  prepared  reagents  should  be  kept  in  glass  vessels.  For 
many,  a  common  medicine  bottle  closed  with  a  cork  will  be 


FIG.  125. 


FIG.  126. 


perfectly  suitable,  though  it  would  be  better  to  use  a  glass-stop- 
pered bottle  whose  stopple  is  ground  to  fit  air-tight.  The  hand- 
iest are  those  whose  stopple  is  prolonged  into  a  long  point  which 
dips  into  the  fluid  and  is  very  convenient  in  taking  out  the 
required  drop  of  the  reagent,  Fig.  125.  For  all  such  reagents 
as  at  the  common  temperature  produce  vapors  which  would  be 
likely  to  harm  the  glass  lenses  of  the  microscope,  or  the  worker 


PREPARATION  OF  REAGENTS.  273 

himself,  a  glass-stoppered  vessel  with  a  double  inclosure  should 
be  employed,  Fig.  126.  It  has  a  glass  stopple  with  slender 
prolongation  well  ground  in.  The  upper  part  of  the  vessel  is 
suddenly  drawn  in  and  a  glass  bell  is  fitted  on  over  the  contrac- 
tion. The  under  edge  of  the  bell  and  the  contracted  part  of  the 
bottle  are  ground  to  fit  each  other  so  that  the  bell  hermetically 
closes  the  upper  part  of  the  flask  outwardly.  These  bottles 
are  especially  recommended  for  ammonia,  ether,  muriatic,  nitric, 
sulphuric  and  acetic  acids. 

The  necessary  manipulations  in  the  preparation  of  the  reagents 
are,  weighing  solid  substances  and  fluids,  measuring  fluids, 
pulverizing  solid  substances,  and  heating,  distilling  and  filter- 
ing fluids.  Pulverizing  solids  should  be  done  in  a  porcelain 
mortar.  For  heating  or  distilling  use,  according  to  the  nature 
and  quantity  of  the  fluid  employed,  a  test  tube,  a  glass  alembic, 
a  small  retort,  watch-glass  or  porcelain  saucer,  which  may  be 
set  during  the  process  on  the  wire  netting  stretched  over  the 
tripod,  or  upon  a  retort  holder.  As  a  source  of  heat  use  a  spirit 
lamp  or  a  Bunsen  gas  burner.  The  operation  of  filtering 
deserves  special  care  and  attention  because  if  one  use  an  impure 
filter,  he  is  likely  to  transfer  a  small  quantity  of  foreign  matter 
to  his  reagent,  which  possibly  may  disastrously  interfere  with 
the  reaction.  The  filter  should  be  prepared  by  putting  a 
considerable  number  of  good  filter  papers  in  a  glass  cup  and 
pouring  upon  them  pure  dilute  hydrochloric  acid,  letting  them 
remain  in  the  same  from  thirty  to  sixty  minutes  ;  then  wash  with 
distilled  water,  as  long  as  litmus  paper  shows  any  acid  reaction 
and  dry  with  moderate  heat.  By  the  use  of  these  purified  filters 
we  are  sufficiently  insured  against  contaminating  the  reagent  in 
the  act  of  filtering.  Many  substances,  like  a  solution  of  caustic 
potash,  mineral  acid,  etc.,  cannot  be  filtered  through  paper  but 
must  be  filtered  through  glass  wool.  For  this  purpose  put  a 
small  quantity  of  the  glass  wool  which  has  been  carefully  washed 
and  dried  in  the  bottom  of  a  small  funnel  and  pour  on  the  fluid. 
Glass  wool  is  much  to  be  preferred  to  asbestos  which  was  form- 
erly used.3 

It  is  often  not  very  easy  to  determine  if  the  substances  used 

3  Asbestos  wool  will  answer,  however,  when  the  glass  cannot  be  had.    A,  B.  H. 
18 


274  THE  MICROSCOPE  IN  BOTANY. 

are  in  truth  chemically  pure,  that  is  if  they  contain  no  admixture 
of  foreign  matter  which  might  make  them  unsuitable  for  use  as 
microscopical  reagents.  In  this  matter  it  is  evident  that  quali- 
tative analysis  can  give  the  only  safe  information,  and  reference 
must  be  made  to  the  numerous  works  and  guides  which  treat 
of  this  subject.  Also  the  methods  for  purifying  the  sub- 
stances to  be  used  (excepting  the  cases  to  be  described  farther 
on)  may  be  looked  up  in  chemical  works. 

For  the  ordinary  purposes  of  the  microscopist  an  apothecary's 
scales  are  fully  sufficient  for  determining  the  parts  by  weight  of 
solid  substances  and  of  fluids.  If  it  be  well  constructed  it  allows 
weighing  from  1  to  2  mg.,  and  this  exactness  is  quite  sufficient. 
In  order  to  prevent  contamination  in  the  process  of  weighing, 
solid  substances  should  never  be  weighed  on  the  open  scale  of 
the  balance  but  on  a  watch-glass  which  has  been  previously 
cleansed  with  care.  The  weight  of  the  glass  can  be  determined 
once  for  all  and  marked  on  the  glass  with  a  writing  diamond. 

The  determination  of  the  parts  of  space  or  volume  of  fluids 
is  done  with  graduated  glass  flasks,  the  so-called  measuring 
flasks.  The  forms  of  glasses  especially  commended  for  the  use 
of  the  microscopist  are  the  following  : 

The  Measuring  Flask.  (Fig.  127.)  This  apparatus  is  com- 
monly a  glass  retort  with  a  long 
narrow  neck,  with  a  mark  on  its 
lower  third.  The  vessel  is  filled  up 
to  this  mark  with  a  fluid  which  is  then 
poured  out  into  another  vessel  and 
measured  to  the  last  drop.  The  cubic 
centimeters  which  it  measures  are 
then  etched  upon  the  side  of  the  flask 
and  become  its  "signature."  It  is 
advised  to  have  several  of  them 
holding  respectively  100,  150,  200, 
FIG.  127.  250,  and  1000  cc.  Smaller  quantities 

of  fluid  are  measured  out  by  means  of  the  well-known 
"pipette,"  two  useful  forms  of  which  are  given  in  Fig.  128. 
Draw  up  the  fluid  in  them  with  the  mouth  till  it  stands  above 
the  mark.  Then  close  the  upper  end  of  the  pipette  with 


THE  BULB  BURETTE. 


275 


the  damp  index  finger  and  carefully  let  as  much  of  the  fluid 
run  back  as  will  bring  it  down  exactly  to  the  mark,  after 
which  empty  the  measured  contents  into  the  vessel  intended 
for  it.  Finally,  put  the  end  of  the  pipette  from  which  the 
fluid  flows  against  the  side  of  the  vessel,  but  without  blowing 
in  it,  and  the  vessel  will  contain  exactly  the  quantity  of  fluid 
indicated  in  the  signature  of  the  pipette.  Pipettes  of  5,  10, 
and  25  cc.  capacity  are  sufficient. 

Still  smaller  quantities   of  fluid  may  be   measured   by  the 
measuring   cylinder,    a   small 
form  of  which  is  given  in  Fig. 
129.       Suppose   we    were   to 
measure  out  1.5  cc.  of  water. 
Fill  up  the  vessel  near  to  the 
desired  quantity.     Let  a  part 
run  out  till  the  surface  stands 
at    some  one  of  the  dividing 
lines,    and   now  pour  slowly, 
drop  by  drop  at  last,   till  the 
fluid  exactly  reaches  the  mark 
1.5  cc.  on  the  cylinder  when 
it  is  held  to  the  light  perpen- 
dicularly before  the  eye.    One 
may  employ  the  siphon  illus- 
trated in  Fig.  75,  p.  162,  for 
taking  up  small    quantities  of 
fluid.     It  should,  however,  be 
previously    moistened    within       FIG.  123. 
and  without  with  the  fluid  to  be  used.. 

For  many  purposes  requiring  very  exact  measuring  the  so-called 
"Burette"  is  indispensable.  The  two  most  serviceable  forms 
are  the  "bulb-burette,"  made  on  the  principle  ofGuy-Lussac 
and  modified  by  Mohr,  and  Mohr's  "spring  compressor  burette." 
Both  forms  are  illustrated  in  Fig.  130. 

The  Bulb  Burette  consists  of  a  somewhat  thick  walled  glass 
tube,  6,  about  400  mm.  long  which  is  rounded  at  the  lower'end 
and  fitted  into  a  wooden  foot,  «,  so  as  to  stand  perpendicularly. 
Its  upper  end  is  closed  with  a  cork  in  which  are  bored  two  holes 


FIG.  129. 


276 


THE  MICROSCOPE  IN  BOTANY. 


into  which  are  fitted  two  glass  tubes.  The  one,  c,  angularly 
bent,  extends  its  long  limb  almost  to  the  bottom  of  the  burette. 
The  tube,  d,  is  less  acutely  bent  and  extends  but  a  little  way  below 
the  cork,  while  upon  the  upper  end  is  fixed  a  rubber  ball  having 
an  opening  on  the  side  which  can  be  closed  with  the  finger. 
When  the  burette  is  filled  with  fluid,  a  certain  quantity  of  it 
may  be  forced  out  through  c,  by  pressing  on  the  bulb  while  the 
thumb  closes  the  orifice.  The  rapidity>of  the  outflow  may  be 
perfectly  regulated  by  the  pressure  upon  the  bulb.  The  bur- 


ette is  filled  by  dipping  the  end  of  the  tube,  c,  in  the  fluid  and 
sucking  at  the  opening  in  e.  The  tube,  b,  has  the  graduation 
in  cubic  centimeters  (holding  from  40  to  60)  and  fractions  of 
the  same  to  about  0.2.  The  value  refers  to  the  interior  diameter 
of  5,  and  the  tube  c,  which  dips  into  the  fluid. 


MOHR'S  SPRING  COMPRESSOR  BURETTE.  277 

Mohr's  Spring  Compressor  Burette  consists  of  a  long  tube,  7^, 
divided  into  whole  and  fifths  of  a  cc.,  open  above  at  i,  and  below 
at  k.  The  lower  end  is  much  narrowed  and  a  piece  of  rubber  tube 
about  27  mm.  long  is  drawn  over  it.  This  is  provided  with  a 
small  glass  tube  having  its  point  drawn  out  as  at  L  The  rubber 
tube  is  closed  by  means  of  a  spring  compressor,  k,  and  the 
burette  is  supported  at  g,  on  the  stand,/,  in  a  perpendicular 
position.  The  tube  is  filled  by  means  of  a  small  funnel  and  the 
opening  at  top  is  afterwards  closed  by  a  small  glass  globe,  with 
a  peg  upon  it,  which  excludes  the  dust.  The  outflow  of  the 
fluid  to  be  measured  is  effected  by  partly  opening  the  spring 
compressor. 

Mohr's  spring  compressor  burette  is  used  chiefly  with  those 
indifferent  fluids  which  would  not  affect  the  rubber  tube,  while 
those  which  would  damage  it  should  be  put  into  the  bulb 
burette  where  they  will  come  in  contact  only  with  glass.4 

The  measuring  vessels  here  described  allow  us,  first,  to 
measure  any  desired  volume  of  fluid,  but  they  can  also,  secondly, 
be  applied  to  measure  quantities  of  fluids  by  weight  when  they 
have  been  changed  into  equivalents  in  volume. 

Take  water  for  instance.  It  is  known  that  1  cc.  of  water,  at 
4°  C.,  weighs  1  g.  So  if  one  has  30.5  cc.  to  weigh  out  he  would 
get  the  right  weight  by  measuring,  at  4°  C.,  30.5  cc.  of  water. 
If  he  has  to  perform  the  operation  at  the  common  temperature 
of  the  room  (say  17°  C.)  then  the  30.5  cc.  of  water  would 
weigh  somewhat  less  than  30.5  g.,  since  the  water  has  a  less 
degree  of  density  at  17°  than  at  4°,  but  this  difference  is  so 
inconsiderable  that  it  may  be  disregarded  in  our  work.  Accord- 
ing to  the  investigations  of  Despretz,  if  the  volume  of  a  gram 
of  water  at  4°  C.  be  1  cc.  at  17°  C.  it  will  be  1.00120  cc. 
Hence  the  volume  of  30.5  g.  at  17°  C.  would  be  30.5366  cc. 
But  the  excess  of  0.036  cc.  is  far  less  than  the  error  one  would 
make  in  reading  off  the  scale. 

4  In  regard  to  getting  the  caliber  of  measuring  vessels,  cf.  Bunsen,  gasometric  method 
(Brunswick,  1857),  pp.  26-38.  Concerning  testing  and  correcting  the  same,  cf.  F.  Mohr,  Lehrb. 
d.  Chem-analyt.,  Volumetric  Method  (4  Aufl.,  Bi  swg.,  1874).  pp.  1-50.  further,  Fresenius 
Anleit.  z.  quantitative  chem.  analyse  (6  Aufl.,  Brschwg.,  1875),  pp.  36-46.  In  the  last  two 
books  are  exact  directions  concerning  all  quantitative  analytical  determinations  ami  the 
cautions  to  be  observed  in  reading  off  the  same. 


278  THE   MICROSCOPE   IN  BOTANY. 

It  is  clear,  therefore,  that  quantities  by  weight,  of  other  fluids, 
may  be  converted  into  equivalents  of  volume  when  we  know 
their  specific  gravity.  If,  for  example,  one  has  30  g.  of  gly- 
cerine to  weigh  out  and  knowing  that  the  specific  gravity  of 
glycerine  is  1.264,  he  needs  to  divide  1.264  by  30  to  get  the  num- 
ber of  cc.  of  glycerine  which  will  weigh  30  g.  The  division 
gives  23.7  cc.  The  following  table  will  be  useful  in  the  re- 
duction of  weight  measures  to  equivalents  of  volume,  for  some 
of  the  fluids  used  as  microscopical  reagents. 

SPECIFIC    GRAVITY   OF   FLUIDS    AT    15°  C. 

Ethyl  ether  0.736  Ammonia  saturated  0.884 

Alcohol  absolute     0.794  Carbolic  acid  "  1.066 

"  90  per  cent         0.823  Chloroform  1.480 

"  50  per  cent         0.919  Acetic  acid  1.055 

"  40  per  cent         0.940  Glycerine  1.264 

Nitric  acid  1.526  Hydrochloric  acid  1.210 

Carbon  disulphide  1.271  Sulphuric  acid  1.842 

Turpentine  oil    0.870 

1   Small  variations  of  the  temperature  of  the  room  may  be  en- 
tirely disregarded  in  the  calculations. 


III.     APPLICATION  OF  THE  VOLUMETRIC  METHOD 

IN  THE  PREPARATION  OF   MICROSCOPICAL 

REAGENTS. 

The  volumetric5  method  may  be  successfully  employed  in  the 
rapid  preparation  of  certain  reagents  as  Frey6  has  rightly  as- 
serted, and,  on  the  other  hand,  it  offers  the  possibility  of  deter- 
mining with  ease  the  per  cent  value  of  certain  simple  fluid 
reagents.  We  may  here  briefly  set  forth  the  few  cases  which 
concern  the  microscopist  from  the  wide  field  of  quantitative 
analysis.  For  the  rest  he  may  consult  the  work  of  Mohr  al- 
ready cited  on  page  277. 

1.  Standard  solution  of  oxalic  acid.  Good  commercial 
oxalic  acid  is  pulverized  and  dissolved  in  a  little  warm  water, 

5  Mohr,  I.  c. 

6  Frey,  Mikroskop,  p.  90. 


STANDARD  POTASSIUM  SOLUTION.  279 

so  that  still  a  considerable  part  of  the  acid  remains  behind  on 
the  bottom  of  the  vessel.  Then  filter  and  crystallize  by  rapid 
cooling.  The  crystals  should  be  drained  ofi*  in  a  filter  and  dried 
at  common  temperature  between  blotting  papers.  To  test  their 
dryness  press  a  piece  of  smooth  paper  upon  them.  If  they  are 
perfectly  dry  none  of  them  will  stick  to  the  paper.7 

Oxalic  acid  which  has  two  molecules  of  water  bound  up  with 
it  has  the  chemical  formula  :  C  O  .  OH 

|  +  2H20 

CO.  OH 

The  atomic  weight  is  126  (C=12,  O  =  16,  H—  1).  Oxalic 
acid  combines  with  two  molecules  of  potassium  hydroxide  H  K  O 
to  form  potassium  oxalate,  C2  O4  K2  +  H2  O,  a  salt  which  contains 
one  molecule  of  water,  after  this  formula  : 


. 
( 

V 


CO.  OH 

| 
CO.OH 


CO.  OK 

+H20)  +  3H20 


C  O.  O  K 

Oxalic  acid  -f-  2  potassium  hydroxide  =  potassium  oxalate 
+  3  water.  To  form  this  salt  the  two  molecules  of  potassium 
hydroxide,  K2  O,  contributes  what  corresponds  to  an  equivalent 
of  94  (K  =  39,  O=16).  If  now  we  reduce  both  numbers 
(126  and  94)  to  one  atom  of  potassium  by  dividing  by  2  we 
shall  have  the  respective  numbers  63  and  47. 

We  now  weigh  out  6.3  g.  of  pure  oxalic  acid  on  the  chemical 
scales  and  dissolve  it  in  exactly  100  cc.  of  distilled  water.  Each 
cubic  centimeter  of  the  solution  will  contain  0.063  g.  of  oxalic 
acid.  With  0.047  g.  of  potassium  added  to  each  cubic  centi- 
meter of  the  oxalic  solution  we  shall  be  able  to  transform  it  to 
potassium  oxalate. 

2.  Standard  potassium  solution.  It  is  necessary  also  to  have 
a  standard  solution  of  potassium,  that  is,  a  solution  of  caustic 
potash  in  water,  which  contains  in  each  cc.  of  the  fluid  0.047  <r. 
of  the  potassium  dissolved.  For  this  purpose  prepare  a  pretty 

7  Fresenius,  1.  c.,  p.  131,  /.—  Mohr,  I.  c.,  p.  81,/. 


280  THE   MICROSCOPE  IN  BOTANY. 

concentrated  solution  of  caustic  potash  in  water,  as  it  will  be 
more  particularly  described  under  the  title  "  potassium  hydrox- 
ide." Transfer  with  the  pipette  any  desired  number  of  cubic 
centimeters  of  the  fluid  to  a  glass  beaker  and  dilute  with  any 
desirable  quantity  of  water.  To  this  fluid  add  a  few  drops  of 
litmus  tincture  till  it  appears  evenly  but  distinctly  blue.8  Then 
let  a  standard  solution  of  oxalic  acid  flow  into  it  from  a  burette 
till  the  litmus  tincture  is  uniformly  changed  to  red.  Now, 
suppose  that  in  order  to  neutralize  5.  cc.  of  our  potash  solution 
we  should  need  to  use  20.5  cc.  of  the  oxalic  acid  solution. 
These  20.5  cc.  contain  20.5  X  0.063  g.  of  oxalic  acid  which 
corresponds  to  20.5  X  0.047  g.  of  potassium.  This  is  con- 
tained in  my  5.  cc.  of  potassium  solution.  If  I  now  dilute 
every  5.  cc.  of  my  solution  of  potash  to  20.5  cc.,  every  cc.  of 
the  solution  will  contain  0.047  g.  of  potassium  and  be  a  standard 
solution.  I  should  add  to  each  of  my  5.  cc.  of  potash  so- 
lution 15.5  cc.  of  distilled  water,  or  what  is  the  same  thing 
put  into  a  100  cc.  flask  32.25  cc.  of  my  potash  solution,  and 
nil  up  to  100  cc.  with  water.  In  regard  to  keeping  solutions 
of  potassium  see  below  under  "  potassium  hydroxide." 

3 .  Standard  solution  of  sulphuric  add.  The  potassium  equiv- 
alent of  0.047  corresponds  to  a  sulphuric  acid  equivalent  of 
0.040  for 

H2  S  O4  +  2  H  KO     =  K2  S  O4      +2  H2  O. 

Sulphuric  acid.      2  Pot.  hydroxide.    Potass,  sulphate.       2  Water. 

SO3'—SO  (S  =  32,O  =  16)  reduced  to  one  atom  of  potas- 
sium equals  40.  Let  5.  cc.  of  pure  English  acid  be  put  in  a 
glass  and  diluted  at  will  and  colored  with  some  drops  of  litmus 
tincture.  Add  now  from  the  burette  as  much  standard  potash 
solution  as  will  change  the  color  to  blue ;  read  off,  and  dilute 
each  cc.  of  the  acid  with  as  many  cc.  of  distilled  water  as  the 
number  of  cc.  of  potash  solution  used.  One  may  employ  the 

s  Digest  for  a  long  time  in  the  water  bath,  1  part  commercial  litmus  with  6  parts  water, 
filter,  divide  tiie  blue  fluid  into  2  parts,  neutralize  the  free  alkali  in  the  one-half,  by  repeat- 
edly stirring  it  with  a  glass  rod  which  has  been  dipped  in  very  dilute  nitric  acid  till  the 
color  appears  red.  Mix  in  then  the  other  blue  half  and  add  one  part  strong  alcohol  and 
keep  the  tincture  thus  prepared  in  an  open  glass  vessel,  in  some  place  free  from  dust.  The 
tincture  loses  its  color  when  kept  in  a  closed  vessel. 


EQUIVALENTS. 


281 


standard  sulphuric  acid  solution  in  place  of  the  oxalic  acid  the 
purifying  of  which  consumes  so  much  time. 

It  is  necessary  to  know  the  equivalent  number  of  the  sub- 
stances used  ia  order  to  employ  these  standard  solutions  in  pre- 
paring microscopical  reagents. 


Sodium 

Potassium 

Ammonia 

Chromic  acid  (Anhydrate)    0.05024 

Tartaric  acid 

Calcium  chlorate 


EQUIVALENTS. 

A.     For  Standard  Solutions. 

0.03100    Barium  chlorate  (Anhydrate)       0.10405 


0.04711     Hydrochloric  acid  0.03G46 

0.01700    Nitric  acid  0.05400 

Acetic  acid  0.06000 

0.07500     Oxalic  acid  0.06300 

0.05546     Snip,  acid  (Anhydrate)  0.04000 


B.     For  -i-  Standard  Solution. 


Potassium  iodide 
Sodium  chloride 


0.016611    Potas.  chloride 
0.005846     Silver 


0.007457 
0.010797 


The  application  of  standard  solutions  to  the  preparation  of 
microscopical  reagents  may  be  illustrated  by  some  practical 
examples. 

Examples  for  the  application  of  the  volumetric  method  in 
preparing  microscopical  reagents. 

1.  To  find  the  percentage  of  a  given  solution  of  potassium. 
Transfer  with  the  pipette  10  cc.  of  the  solution  to  a  glass  cup, 
dilute  with  distilled  water,  add  litmus  tincture,  and  Jet  standard 
sulphuric  acid  solution  flow  in  till  the  color  changes.  It  requires 
75.5  g. 

1  cc.  acid  =  0.047  g.  alkali 
75.5  cc.  "  =3.5485  g.    " 

This  3.5485  g.  of  potassium  is  contained  in  10  cc.  of  water. 
In  100  cc.  (100  g.)  there  would  be  35.485  g.  The  solution 
then  contains  35.485  per  cent  of  potash. 


282  THE  MICROSCOPE  IN  BOTANY. 

2.  To  prepare  a  solution  of  a  definite  solution  of  potash,  say 
33.3  per  cent.     Put  5.  cc.  of  a  partly  concentrated  solution  of 
potassium  in  a  glass,  dilute  with  water,  color  with  litmus,  and 
add  the  necessary  quantity  of  standard  sulphuric  acid  solution 
=  58.4  cc. 

1  cc.  acid  =  0.047  g.  alkali 

58.4   "     =2. 7448  g.    "      in  5  cc.  solution 
Percentage  =  2.7448  X  20  =  54.9 
The  proportion  33.3  :  100  : :  54.9  :  x 

x=  164.86 

Therefore  100  cc.  of  our  solution  must  be  diluted  to  164.86, 
or  10  cc.  to  16.486  cc.  to  give  us  a  33.3  per  cent  solution. 

3.  To  prepare  a  1  per  cent  solution  of  acetic  acid.     Take 
10  cc.  of  dilute  acetic  acid.     Dilute  with  water  and  add  litmus 
tincture.     The  necessary  potassium  solution  for   neutralization 
is  32.  cc. 

1  cc.  potassium  =  0.06  g.  acetic  acid 
32  cc.       "        =1.92         "         " 
1.92  X  10  =  19.2  =  percentage 
1:100::  19. 2:o:     a  =1920 

To  get  1  per  cent  solution,  therefore,  we  must  dilute  100  cc. 
of  our  tested  solution  to  1920  cc.,  or  1  cc.  to  19.20  cc. 

4.  English  sulphuric  acid  of  unknown  strength  to  be  diluted 
to  a  20  per  cent  solution.     1  cc.  of  acid  requires  23.2  cc.  of 
standard  potassium  solution  for  neutralization. 

1  cc.  potassium  =  0.04  g.  sulp.  acid 
23.2         "         =  0.928g. 
0.928  X  100  =  92.8  =  percentage 
20  :  100  : :  92.8  :  x,     x  =  464 

So  we  must  dilute  100  cc.  of  the  acid  to  464  cc.,  or  10  cc. 
to  46.4  cc.  to  get  our  20  per  cent  solution. 

5.  To  test  the  correctness  of  a  5  per  cent  solution  of  chro- 
mic acid.     Chromic  acid  to    be  used   as    a  microscopical  rea- 
gent should  have  no  foreign  admixture.     Most  of  all  should  it 
have  no  trace  of  sulphuric  acid.     Consequently  its  volumetric 
analysis  should  be  accomplished  by  means  of  a  barium  chloride 


INORGANIC  COMBINATIONS.  283 

solution.  Make  a  standard  solution  of  barium  chloride  (Ba  C12 
+  2  H2  O)  by  dissolving  12.2  g.  of  the  crystals  in  100  cc.  of 
water,  1  cc.  of  this  standard  solution  corresponds  to  0.05024  g. 
of  chromic  acid.  Put  25  cc.  of  the  chromic  acid  solution  to  be 
tested  in  a  glass,  add  a  few  drops  of  concentrated  acetic  acid 
and  boil.  Then  add  fluid  ammonia  which  is  free  from  carbonic 
acid  till  it  feebly  predominates  and  then  heat.  A  Guy-Lussac 
bulb  burette  is  filled  with  the  standard  solution  of  the  barium 
chloride,  and  added  drop  by  drop  to  the  fluid  which  is  being 
tested,  that  meanwhile  being  constantly  shaken.  There  is 
produced  a  bright  yellow  precipitate,  insoluble  in  hot  water. 
The  addition  of  the  normal  solution  should  cease  when  the 
yellow  color  of  the  fluid  begins  to  grow  indistinct.  The  reac- 
tion is  ended  at  the  moment  when  the  yellow  color  disappears 
from  the  solution.  It  required  25.5  cc.  of  the  barium  chloride. 

1  cc.  of  barium  chloride  =  0.05024  g.  chromic  acid 
25.5  cc         "         "          =  1.28  g.  chromic  acid 
25  :  1.28  : :  100  :  x    x  =  5.12  =  per  cent. 

The  solution  is  something  more  than  5  per  cent.  If  an  ex- 
actly 5  per  cent  solution  is  to  be  made  it  must  be  by  the  pro- 
portion 

5:  100::  5. 12:  x 
x  =102.4 

So  100  cc.  of  the  solution  must  be  diluted  to  102.4  cc. 


IV.     ENUMERATION  AND  PREPARATION  OF 
MICROSCOPICAL  REAGENTS. 

A.     INORGANIC  COMBINATIONS. 

1.    WATER     H2O. 

It  is  always  understood  that  the  water  used  by  the  microscop- 
ist  is  distilled.  It  may  be  prepared  in  the  well-known  way  or 
purchased  of  the  druggist.  In  the  latter  case  it  should  be  care- 
fully filtered  before  using.  Potassium  oxalate  gives  no  trace 


284  THE  MICROSCOPE  IN  BOTANY. 

of  a  precipitate  in  pure  distilled  water  even  after  long  action, 
showing  the  absence  of  calcium  salts.  Should  it  be  desired  to 
use  water  free  from  carbonic  acid,  it  should  be  boiled  immedi- 
ately before  using. 

2.    NITRIC  ACID.    HNO3. 

One  should  use  the  pure  acid  of  the  pharmacopoeia  which  is 
perfectly  colorless,  has  a  stinging  odor  and  develops  a  slight 
vapor  in  the  air.  It  is  used  in  a  concentrated  form,  as  well  as 
diluted  with  water  in  various  proportions  as,  for  example,  50, 
SO,  10  per  cent.  The  dilution  may  easily  be  made  by  the  volu- 
metric method.  This,  and  the  next  following  acids,  should  be 
kept  in  double-stoppered  bottles  (Fig.  126,  p.  272),  and  on 
account  of  their  development  of  fumes  should  be  used  under 
the  microscope  only  with  the  largest  cover-glasses. 

Nitric  acid  is  used  as  a  medium  for  maceration  with  or  without 
potassium  chlorate  (see  p.  163),  and  with  ammonia  for  reactions 
on  nitrogenous  and  corky  substances  and  middle  lamella. 

3.     SULPHUBIC  ACID.    H2  S  O4. 

Likewise  only  the  pure,  the  so-called  English  acid,  should  be 
used  either  in  a  concentrated  state,  or  in  different  dilutions  with 
water,  1  volume  of  acid  to  3  of  water  or  4  of  water,  specific 
gravity  1.20  or  with  greater  quantities  of  water.  It  is  used 
for  dissolving  cell  membranes  and  cell  contents  ;  with  iodine  ten- 
test  ing  cellulose,  and  with  indol  for  membranes  of  wood  cells. 

4.     HYDROCHLORIC  ACID.    H  Cl. 
(Muriatic  Acid.) 

As  with  the  foregoing  this  acid  should  be  employed  only  in 
the  pure  state,  as  it  is  used  by  the  apothecary.  It  is  perfectly 
colorless.  The  yellow  tinted  has  been  contaminated  by  com- 
bination with  iron.  It  is  used  either  concentrated,  or  in  various 
dilutions.  In  the  first  case  (cold  saturated)  it  fumes  in  the 
air  and  like  nitric  acid  should  be  used  only  with  the  largest 


INORGANIC  COMBINATIONS.  285 

cover-glasses.  By  heating  the  concentrated  acid  to  the  boiling 
point  (110°),  a  20.2  per  cent  acid  is  produced  which  does  not 
fume,  and  is  recommended,  for  many  microscopical  purposes,  as 
it  has  not  been  heretofore,  to  my  knowledge.  This  acid  does 
not  affect  the  lenses  in  the  least  and  should  be  used  in  all  cases 
where  the  concentrated  is  not  necessary. 

Hydrochloric  acid  is  a  macerating  and  decalcifying  medium 
(see  p.  164),  and  serves  as  a  test  for  proteids  and  calcium  car- 
bonate crystals  and  others. 

5.    PHOSPHORIC  ACID     HPO3. 
(Metaphosphoric  Acid.) 

Forms  an  icy  looking,  glassy,  solid  substance  (Acidum  phos- 
phoricum  glaciate)  which  dissolves  to  a  colorless%fluid  in  water. 
As  a  microscopical  reagent  it  is  very  little  used. 

6.    SOLUTIONS  OF  IODINE. 

In  botanic  microscopical  analysis,  solutions  of  iodine  are  the 
reagents  most  frequently  used.  They  are  solutions  of  pure 
iodine9  in  different  fluids,  water,  alcohol,  glycerine,  solutions  of 
potassium  iodide  and  of  zinc  chloride.  They  bear  the  names 
iodine  water,  iodine  alcohol,  iodine  glycerine,  potassium  iodide 
of  iodine,  and  chlor-iodide  of  zinc.  The  methods  of  preparing 
these  various  reagents  are  as  follows.10 

1.  Iodine  water.  Iodine  is  soluble  in  water  only  in  a  very 
small  quantity  (1  :  0.00014)  and  forms  a  slightly  brownish- 
yellow  colored  fluid.  It  is  prepared  by  putting  a  small  fragment 
in  distilled  water,  the  solution  taking  place  in  the  course  of  a 
few  days.  A  still  better  way  is  to  prepare  the  solution  imme- 
diately on  the  slide,  by  putting  a  small  splinter  of  iodine  in  the 

9  For  ordinary  cases  the  crystalline  commercial  iodine  will  do.    But  if  one  prefers  that 
which  contains  no  trace  of  bromine  he  may  prepare  it  by  putting  pulverized  potassium 
iodide  mixed  with  black  oxide  of  magnesia  in  a  small  retort,  and  pour  sulphuric  acid 
over  it.    When  the  mixture  is  heated  the  iodine  distils  over  and  collects  either  in  the  neck 
of  the  retort  or  in  a  receiver. 

10  Harting,  Das  Mikroskop,  p.  424,  475,  /.,  499  — Harting.  Entwicklungsgesch.  des  Pflan- 
zenkeims,  p.  35,  153,  /.—  Nageli  u.  Sch\vendener,  Das  Mikroskop,  p.  473,  /.—  Dippel,  Das 
Mikroskop,  Bd.  I,  p.  273  ff.~  Frey,  Das  Mikroskop,  p,  82,  /.— Ponlsen,  BotauiskMikrokemi, 
P-  1,  ff. 


286  THE  MICROSCOPE  IN  BOTANY. 

water  with  the  preparation  under  the  cover-glass.  The  reagent 
works  with  more  certainty  when  it  is  prepared  immediately 
before  using. 

2.  Iodine  alcohol.     Iodine  dissolves  readily  in  absolute  al- 
cohol and  produces  a  deep  brown  fluid  known  under  the  name 
"tincture  of  iodine."     For  microscopical  purposes  we  make  the 
solution  with  a  little  excess  of  iodine,  and  afterwards  dilute  with 
absolute   alcohol  or   distilled  water.     In  the  latter  case  some 
iodine  will  be  separated.     It  is  best  to  prepare  it  shortly  before 
using. 

3.  Glycerine  iodine.     There  are  two  modifications  of  this 
reagent  in  use. 

(a)  Pure  concentrated  glycerine  to  which  is  added  a  sufficient 
quantity  of  iodine,  which  dissolves  slowly  but  abundantly  and 
communicates  a  beautiful  red  brown  color  to  the  fluid.     This 
may  be  used  either  in  a  concentrated  state,  or  in  different  dilu- 
tions.    The  diluting  medium  may  be  either  water  or  glycerine — 
the  latter  in  case  the  object  will  bear  the  addition  of  no  water. 

(b)  Dissolve,  according  to  requirement,  a  greater  or  less  quan- 
tity of  potassium  iodide  in  glycerine,  and  add  to  this  solution, 
metallic  iodine. 

4.  Potassium  iudide  of  iodine.     Three  grammes  of  crystal- 
lized potassium  iodide  is  dissolved  in  60  cc.  of  distilled  water 
and  to  this  solution  is  added  1  gramme  of  metallic  iodine.     The 
resulting  solution  has  a  dark  brown  red  color  and  can  be  diluted 
to  suit  by  the  addition  of  distilled  water.     For  the  investigation 
of  certain  objects,  as  lichen  utricle,11  the  following  composition 
is    recommended.      Iodine  0.06  g.,  potassium    iodide    0.2  g., 
distilled  water  16  g. 

5.  Chlor-iodide  of  zinc.     Pure  zinc  in  rods  as  it  is  used  in 
the  chemical  laboratory  is  dissolved  to  saturation  in  muriatic  acid. 
Still  more  zinc  is  added  and  the  solution  evaporated  till  it  be- 
comes a  thick  fluid.     In  this  concentrated  solution  of  zinc  chlor- 
ide dissolve  to  saturation  potassium  iodide,  and  then   add   a 
considerable  quantity  of  metallic  iodine  which  will  slowly  dis- 
solve in  the  mixture  (Schultz).     Radlkofer12  has  given  a  more 

11  Poulsen,  1.  c.,  p.  4.    (German  Trans,  p.  6.    English  Trans,  p.  6 .) 

12  Dippel,  I.  c.t  Bd.  I,  y.  274,/. 


INORGANIC  COMBINATIONS.  287 

exact  formula  for  the  preparation  of  chlor-iodide  of  zinc  solution. 
Evaporate  a  solution  of  zinc  chloride  prepared  in  the  common 
temperature  in  a  heat  not  exceeding  that  of  boiling  water,  to  a 
clear  syrup  of  the  specific  weight  of  2.0  and  then  dilute  it  with 
water  to  a  specific  weight  of  1.8  which  will  require  12  parts 
water  to  100  of  the  solution.  In  100  parts  of  this  fluid  dissolve 
by  gentle  heat  6  parts  of  potassium  iodide,  and  as  much  iodine 
as  it  will  take  up.  The  iodine  solution  of  zinc  chloride  has  now 
the  consistency  of  concentrated  sulphuric  acid.  It  is  perfectly 
clear  and  possesses  a  bright  yellow-brown  color. 

Chlor-iodide  of  zinc  solution  —  a  very  important  reagent  — 
may  be  suitably  employed  under  the  following  modifications.13 

(a)  A  concentrated  solution  weak  in  iodine.     The  zinc  solu- 
tion prepared  according  to  Radlkofer's  formula,  concentrated 
and  mixed  with  potassium  iodide  dissolves  in  the  course  of  about 
48  hours  as  much  iodine  as  will  give  it  a  bright  yellow-brown 
color.     This  modification  can  be  used  with  those  preparations 
which  a  stronger  iodine  mixture  would  color  too  intensely. 

(b)  Concentrated  solution  strong  in   iodine.       This    is   pro- 
duced by  keeping  the  solution  for  several  weeks  in  a  dark  place 
with  an  excess  of  metallic  iodine.     In  the  course  of  time  it  takes 
up  so  much  iodine  as  to  become  of  a  dark  red-brown  color, 
holding  a  place    somewhere  about  midway  between    glycerine 
iodine  and  potassium  iodide  of  iodine. 

(c)  Dilute  solutions  may  be  obtained   from  b  by  the  addi- 
tion   of  various  large  quantities  of  potassium  iodide  which  is 
dissolved  in  distilled  water  in  the  proportion  of  1 :  20. 

(d)  For  some  purposes  it  is  recommended  to  mix  the  iodine 
with  the  concentrated  zinc  chloride,  under  the  cover-glass,  with 
the  preparation  which  is  being  examined.     In  that  case  the  zinc 
solution  should  be  used  without  the  potassium  iodide,  and  a 
little  splinter  of  iodine  added  (Nageli) . 

Hy dried ic  acid  is  formed  in  all  iodine  reagents  by  the  influence 
of  light.  There  are  found  traces  of  it,  for  instance,  in  iodine 
water  after  a  few  hours.  It  betrays  its  presence  by  an  acid 
reaction.14  Many  reactions  are  interfered  with  by  the  presence 

is  Behrens  in  Flora,  1879,  p.  239,  in  separate  print,  p.  58. 

"  Nageli  iu  Sitzungsber.  d.  Bayer,  Acad.,  1SG3,  Bd.  I,  p.  4S4. 


288  THE  MICROSCOPE  IN  BOTANY. 

of  hydriodic  acid.  In  iodine  water  and  iodine  alcohol,  which 
especially  should  be  free  from  acid,  its  presence  may  be  de- 
tected by  litmus  paper,  or  a  less  quantity  by  the  following  ex- 
periment. Placing  a  small  quantity  of  starch  on  a  slide,  add 
some  of  the  iodine  to  be  tested  and  let  it  all' dry  up.  Now  if 
the  solution  contains  no  hydriodic  acid  the  starch  which  was 
colored  blue  by  the  iodine  will  keep  its  color.  If  there  is  any 
hydriodic  acid  the  starch  will  become  yellow  by  drying.  If  we 
want  to  use  perfectly  pure  solutions  of  iodine  we  must  freshly 
prepare  them  each  time.  All  solutions  of  iodine  are  to  be  kept 
in  the  dark. 

Iodine  reagents  are  useful  in  testing  starch,  cellulose  and  its 
modifications  as  well  as  the  proteids. 

7.    POTASSIUM  HYDKOXIDE  K  H  O. 
(Caustic  Potash.) 

It  may  be  found  in  the  market  quite  pure  in  snow-white  sticks 
of  a  glassy  crystalline  fracture,  dissolves  in  water  with  great 
eagerness  and  with  the  development  of  considerable  heat.  In 
a  dry  state  the  potassium  acts  quite  indifferently  towards  the 
carbonic  acid  of  the  air,  but  in  damp  air  it  absorbs  carbonic 
acid  and  water  and  is  slowly  transformed  into  potassium  car- 
bonate. The  aqueous  solution  possesses  the  same  quality  in  a 
high  degree. 

The  presence  of  potassium  carbonate  in  a  potassium  solution 
is  easily  detected  by  a  sufficient  addition  of  an  acid,  effervesc- 
ence taking  place  in  that  case.  In  a  potassium  solution  con- 
taining carbonic  acid,  baryta  water  produces  a  white  precipitate, 
barium  carbonate.  As  the  presence  of  potassium  carbonate 
works  disastrously  in  many  microscopical  reagents  the  solu- 
tion should  be  freshly  prepared  whenever  it  is  to  be  used. 
For  this  purpose  put  the  stick  of  potassium  in  a  glass  vessel, 
pour  over  distilled  water  and  let  it  dissolve.  As  the  potassium 
stick  becomes  clearer,  that  is  as  the  layer  of  potassium  carbonate 
is  dissolved  off,  turn  off  the  water  and  replace  it  with  new. 
This  second  solution  is  the  one  to  use.  Keeping  it  but  twenty- 
four  hours  it  will  absorb  carbonic  acid.  Since  the  frequent 


INORGANIC  COMBINATIONS. 


289 


preparation  of  this  solution  is  a  waste  of  both  time  and  money, 
I  have  constructed  the  following  contrivance  in  which  caustic 
potash  solution  may  be  kept  for  months  or  years  without  absorb- 
ing carbonic  acid  from  the  air. 

A  vessel  containing  the  solution  closed  with  a  cork  or  glass- 
stopple  will  not  accomplish  this,  since  every  change  in  the  tem- 
perature will  effect  a  change  in  the  volume  of  air  in  the  vessel 
by  making  it  smaller  or  larger  and  so  will  cause  a  little  of  the 
outside  air  to  find  its  way  within.  But  if  one  allows  the  air 
free  entrance  and  exit  from  the  flask  but  removes  the  carbonic 
acid  from  it  before  it  enters,  the  solution  will  always  remain  free 
from  carbonic  acid.  The  apparatus  illustrated  in  Fig.  131  rests 


n 


FIG.  131. 

upon  this  principle.  The  wide-necked,  clean  and  dry  flask  a 
is  closed  with  a  cork,  &,  perforated  in  two  places,  through  which 
pass  the  tubes,  jfand  c;  /is  a  narrow  glass  tube  which  reaches 
almost  to  the  bottom  of  a.  At  /*,  it  is  bent  almost  to  a  half 
circle  and  drawn  out  to  a  fine  point  at  g  ;  c  is  an  ordinary  straight 
calcium  chloride  tube  closed  at  d,  with  a  rubber  stopper  to  ex- 
clude the  dust,  through  which  is  thrust  the  small  glass  tube  e, 
open  at  both  ends.  The  calcium  chloride  tube  must  previously 

19 


290  THE  MICROSCOPE  IN  BOTANY. 

be  filled  with  a  dry  substance  which  will  absorb  carbonic  acid. 
Graham  has  recommended  for  that  purpose  a  mixture  of  Glauber's 
salts  and  quicklime.  For  the  preparation  of  this,  pound  like 
parts  of  each  in  a  porcelain  mortar.  Let  the  mixture  effervesce 
fully  and  dry  it  in  a  tin  dish  over  a  free  flame.  Into  the  tube 
c,  put  a  lock  of  glass  wool  to  keep  small  pieces  of  the  above 
described  mixture  from  falling  through.  Then  fill  the  tube  with 
the  absorbing  mixture  which  should  not  be  a  powder  but  small 
pieces.  Then  put  a  thin  layer  of  wax  over  the  cork  stopple 
and  over  this  paint  a  coat  of  asphalt  varnish.  After  the  varnish 
is  dry  the  bottle  is  to  be  filled  with  the  solution  of  caustic  potash 
freshly  prepared  by  the  above  described  process.  Before  the 
filling  takes  place  blow  in  at  e  till  the  air  having  carbonic  acid 
in  it  is  all  driven  out.  Then  dip  the  end  of  the  tube  g  in  the 
solution,  and  suck  at  e  till  the  bottle  is  filled.  Then  put  over 
the  end  of  g  a  little  piece  of  thick  walled  rubber  tube  with  a 
piece  of  glass  rod  filling  and  closing  the  end  of  it.  So  arranged, . 
the  apparatus  can  stand  for  months  long  and  not  absorb  the  least 
trace  of  carbonic  acid.  If  I  want  a  drop  of  the  solution  on  the 
slide,  I  remove  the  cap  from  g  and  put  it  over  the  tube  e,  and 
then  placing  my  hand  on  the  part  of  the  flask  «,  which  contains 
the  air,  the  air  will  be  warmed  and  expanded,  and  conse- 
quently will  push  out  through  the  narrow  tube  <7,  the  required 
quantity  of  the  alkali.  Should  it  be  desired  to  transfer  larger 
quantities  of  the  potassium  from  a,  into  another  vessel,  we  may 
connect  it  with  a  common  wash  bottle  which  is  half  full  of  the 
ordinary  solution  of  potash.  Then  remove  the  spring  clips,  I 
and  7i,  shove  m  down  over  e  and  blow  in  o,  till  the  desired 
quantity  of  the  alkali  has  been  driven  out  through  g. 

Potassium  hydroxide  is  used  in  alcoholic  as  well  as  in  aqueous 
solution.  The  preparation  of  the  former  (Russow's  potassium 
alcohol)  has  already  been  described  on  p.  200.  The  aqueous 
solution  is  used  both  in  its  concentrated  and  dilute  forms,  the 
alkaline  element  being  gauged  to  meet  the  resistance  of  the 
particular  object  tested.  The  quantity  of  potassium  in  the  solu- 
tions can  easily  be  determined  by  the  volumetric  method. 

There  are  manifold  uses  for  potash  solutions  in  microscopy. 
Besides  its  use  for  bleaching  (pp.  199,  200),  it  has  a  reaction  for 


INORGANIC  COMBINATIONS.  291 

protoplasm,   cork   substance,   tannic   acid,   crysophauic    acid, 
sugar,  etc.,  etc. 

8.    POTASSIUM  CHLORATE    K  Cl  O3. 

This  is  a  small,  whitish,  greasy-looking  crystal  fouud  suffi- 
ciently pure  in  the  market  and  used  chiefly  in  the  preparation  of 
maceration  mixtures  (see  p.  163). 

9.    POTASSIUM  NITRATE    K  N  O3. 

Its  use  in  botanical  microscopy  is  extremely  limited.  It  is 
procured  pure  most  .easily  by  neutralizing  a  solution  of  potassium 
with  nitric  acid  and  crystallizing  the  resulting  salt. 

10.    POTASSIUM  BICHROMATE    K2O2Or 
(Potassium  pyrochromate.) 

It  can  be  got  sufficiently  pure  by  taking  the  larger  crystals  of 
the  common  salt,  washing  out,  and  recrystallizing  three  or  four 
times.  Dissolved  in  water  it  is  used  for  hardening  tissue  (see 
p.  178),  and  as  a  test  for  gum  and  tannic  acid. 

11.     SODIUM  CHLORIDE    NaCl. 
(Common  Salt.) 

Common  cooking  salt  contains  numerous  foreign  substances. 
Its  preparation  for  our  uses  is  best  done  from  pure  potassium 
carbonate  (or  bicarbonate)  by  mixing  with  hydrochloric  acid 
and  crystallizing.  It  is  used  but  rarely  and  in  very  weak  solu- 
tions. 

12.    AMMONIA    (NH)4HO. 
(Ammonia  hydroxide.) 

This  is  frequently  used  in  saturated  aqueous  or  dilute  solutions. 
It  is  sold  in  the  drug  shops  under  the  name  of  Liquor  nmrnonia 
caustica.  If  one  wishes  to  prepare  it  for  himself  he  iiiay  do  it 
with  sal  ammoniac  and  quicklime,  washing  the  developed  am- 
monia gas  in  a  small  water  flask  in  strong  potash  or  soda  lye, 


292  THE  MICROSCOPE  IN  BOTANY. 

and  leading  thence  in  a  double  bent  tube  to  the  absorption  flask 
filled  with  cold  distilled  water,  carrying  the  end  of  the  tube  down 
very  near  to  the  bottom  of  the  flask.  The  absorption  flask 
should  be  set  in  cold  water.  The  fluid  is  saturated  with  the 
ammonia  when  bubbles  of  the  gas  rise  through  it. 

Ammonia  may  many  times  be  used  in  microscopy  in  place  of 
caustic  potash.  It  comes  into  use  also  in  Hanstein's  method  of 
bleaching  (see  p.  199).  With  nitric  acid  it  is  a  test  for  proteid 
combinations. 

13.    FERRIC  CHLORIDE    Fe  C16 

For  sale  by  the  apothecary  under  the  name  of  Liquor  ferri 
sesquichlorate.  The  aqueous  solution  is  employed  in  micro- 
scopical analysis  in  not  too  dilute  a  state  for  a  test  of  tannic 
acid.  The  combination  can  also  be  obtained Jby  dissolving  iron 
in  nitro-muriatic  acid  (Aqua  regia^I  Vol.,  H  N  O3  +  IV  Vol., 
HC1).  After  evaporation  the  ferric  chlorate  remains  as  a 
greenish  mass  sometimes  also  as  crystals. 

14.     CHROMIC  ACID    CrO3 

(Aiihydrated  chromic  acid.) 

It  can  usually  be  had  sufficiently  pure,  as  beautiful  red  pre- 
cipitate crystals,  in  the  market.  It  easily  dissolves  in  water 
with  brown,  and  in  dilute  state,  yellow  color.  For  microscop- 
ical purposes  chromic  acid  should  contain  no  sulphuric  acid  (see 
p.  282).  It  is  tested  by  the  so-called  Hepar-test.15  Chromic 
acid  is  used  in  solutions  of  various  strength,  1  part  acid  and  6 
parts  water  for  studying  the  la}ersof  starch  grains  (Dippel), 
also  a  one  per  cent  solution  as  a  hardening  medium.  The 
degree  of  concentration  .of  Uhe  chromic  acid  solution  may  be 
determined  by  the  volumetric  method.  The  solid  acid  should 
be  kept  dry  in  a  well  closed  glass  vessel.  • 

15  Of  the  chromic  acid  which  is  supposed  to  contain  sulphuric  acid,  make  a  pretty  strong 
solution  and  add  to  it  baryta  water,  barium  chromate,  and  if  there  be  sulphuric  acid  pres- 
ent barium  sulphate  will  be  precipitated.  Filter  and  wash  the  precipitate  and  put  some  of 
it  on  charcoal  and  melt  it  down  with  soda. 

Put  the  calcined  mass  when  cold  on  a  silver  piece  and  moisten  it.  If  sulphuric  acid  be 
present,  the  silver  surface  will  be  blackened  or  turned  a  dark  yellow  by  the  formation  of 
silver  sulphates  at  the  point  where  the  mass  rests.  » 


INORGANIC  COMBINATIONS.  293 

15.     COPPER  SULPHATE    CuSO4  +  5H,;O 
(Blue  vitriol.) 

A  sufficiently  pure  preparation  for  most  purposes  may  be  made 
by  crystallizing  the  commercial  blue  vitriol  three  or  four  times. 
A  purer  article  will  be  obtained  if  we  add  ammonia*  flu  id  to  a 
concentrated  aqueous  solution  of  this  salt,  from  which  cupric 
hydroxide  will  be  precipitated  (Cu  O,  H2O).  Then  wash  till 
the  wash  water  shows  no  trace  of  cloudiness  when  baryta  water 
is  added.  Then  dissolve  the  precipitate  in  dilute  sulphuric  acid 
and  evaporate  to  crystallization  and  we  have  the  pure  sulphate. 
It  is  used  mostly  in  a  concentrated,  rarely  in  a  dilute,  aqueous 
solution. 

Copper  sulphate  is  used  in  connection  with  potassium  hydr- 
oxide as  a  test  for  cane  and  grape  sugar,  dextrine,  p  rote  id  sub- 
stances, etc. 

16.     CTTPRAMMOBTIA  ,  Cu  2  (N  H4)  O2 
(Copper  oxide  ammonia.     Schweitzer's  Reagent.) 

This  reagent  was  discovered  in  1857  by  Schweitzer.  It  forms 
a  beautiful  blue  fluid  which  easily  decomposes,  especially  in  the 
light,  and  is  produced  by  a  combination  of  ammonia  oxide  and 
copper  oxide.  The  reagent  has  been  prepared  by  different 
methods,  the  more  important  being  the  following. 

Schweitzer16  prepared  the  solution  used  by  him  in  the  following 
way.  "I  prepare  the  basic-hyposulphate  of  copper  oxide  de- 
scribed by  Heereu  by  a  careful  precipitation  of  a  solution  of  hy- 
posulphate  of  copper  oxide  by  means  of  a  dilute  ammonia  fluid, 
filtering  and  washing  the  bright  green  precipitate.  Then  I 
put  this  combination  still  damp  into  concentrated  liquid  am- 
monia*. It  easily  dissolves  with  the  development  of  heat,  but 
on  codling  crystals  of^  hyposulphurous-copper  oxide  ammonia 
are  formed  from  the  solution.  Together  with  this  cupram- 
monia  must  have  been  formed  by  the  dissolving  of  the  basic 
salt  in  ammonia,  and  this  must  be  contained  freely  dissolved 

J6  Journal  fur  prakt.  Chemie,  Bd.  LXXII  (Leipzig,  1857),  p.  109,  ff. 


294  THE  MICROSCOPE  IN  BOTANY. 

in   the  dark   blue   fluid   from  which   the   crystals   have   been 
separated." 

Bottcher17  used  a  glass  tube  about  two  feet  long  and  one  to 
two  inches  wide,  open  at  the  top  and  narrowed  at  the  bottom, 
terminating  in  a  rubber  tube  provided  with  a  spring  clip.  This 
tube  was  loosely  filled  with  strips  of  copper  rolled  thin  nnd 
then  placed  upright  in  a  holder  and  filled  with  strong  ammonia 
fluid.  After  a  few  minutes  this  was  permitted  to  run  off  into  a 
vessel  set  to  catch  it,  and  again  turned  back  over  the  copper  and 
so  on  for  some  hours.  Thus  in  a  relatively  short  time  one  may 
get  a  deep  dark  blue  fluid  perfectly  saturated  with  copper  oxide. 

Neubauer18  used  for  preparation  a  copper  oxide  which  was 
precipitated  by  the  presence  of  sal-ammoniac  from  copper  vitriol 
solution  with  soda  lye.  The  precipitate  should  be  thoroughly 
washed  first  by  decantatiou  and  at  last  by  filtering  and  then  kept 
damp  under  water.  For  the  preparation  of  the  reagent  it  is 
shaken  up  in  an  excess  of  ammonia  as  long  as  any  will  dissolve 
in  it.  The  result  is  a  deep  blue  solution. 

According  to  Wiesner19  the  cuprammonia  solution  is  prepared 
by  turning  13-16  per  cent  ammonia  water  over  a  quantity  of 
copper  chips  in  an  open  flask  and  letting  it  stand. 

I  have  employed  another  method  similar  to  that  of  Neubauer, 
which  gives  a  very  efficient  and  pure  preparation.  If  we  pre- 
cipitate cupric  sulphate  with  caustic  soda  or  potash,  there  will 
commonly  form,  corresponding  to  the  carbonic  acid  contained  in 
the  alkali,  traces  of  basic  cupric  carbonate  (Cu2  C  O5  H2) ,  in  the 
form  of  a  blue  green  precipitate  which  is  soluble  in  ammonia. 
On  the  other  hand  a  cuprammonia  is  more  effective,  for  which 
the  cupric  hydroxide  has  been  precipitated  with  an  ammoniacal 
salt  mixed  with  a  solution  of  cupric  sulphate.  The  next  fol- 
lowing method  of  preparation  prevents  the  first  unfavorable 
result  just  now  named,  and  furnishes  an  intensely  efficient 
reagent.  Dissolve  2  g.  of  quite  pure  crystallized  cupric  sul- 
phate in  100  cc.  of  distilled  water  and  to  the  solution  add  a  few 

17  Bottcher  in  Nenes  Repert.  fur  Pharmacie,  Bd.  XXIII,  p.  732. 

18  C.  Neubauer  in  Zeitschr.  fur  Aualytic..Chemie  von  Fresenius  Jahrg.  XIV(Weisbaden, 
1875),  p.  196. 

19  J.   Wiesner,   Concerning   the   influence   of  cuprammonia   on   animal   tissue   and 
tissue  elements  (Sitzungsber.  d.  math.-uaturw.  Cl.  d.  K.    Acad.  d.  Wiss.,  Wieii,  Bd. 
XLVI1I.    Abth.  II,  1803,  p.  199,  ff). 


INORGANIC  COMBINATIONS.  295 

drops  of  concentrated  chlorammonia  solution.  Then  prepare 
a  weak  solution  of  potash  with  1  g.  of  caustic  potash  to  100  cc. 
of  distilled  water  to  which  add  a  little  baryta  water  by  which 
any  potassium  carbonate  which  is  present  may  be  transformed 
to  barium  carbonate  and  thrown  down  as  a  white  precipitate. 
The  two  solutions  are  then  to  be  poured  together,  and  the  cupric 
hydroxide  will  precipitate,  from  which,  when  it  has  settled, 
the  water  is  to  be  turned  off  and  distilled  water  substituted 
and  this  again  repeated  several  times,  and  finally  filtered. 
The  washing  of  the  precipitate  must  necessarily  go  on  till  the 
wash  water  shows  no  white  precipitate  when  baryta  water  is 
added.  The  still  damp  cupric  hydroxide  is  put  in  a  suitable 
vessel  and  a  little  concentrated  fluid  ammonia  poured  over  it 
till  all  of  the  hydrate  is  dissolved  ;  then,  finally,  filter  through 
glass  wool.  By  this  means  the  barium  salts  insoluble  in  am- 
monia (barium  sulphate  and  carbonate)  are  separated  out  and 
are  left  behind,  also  commonly  a  small  quantity  of  the  cupric 
oxide  (CuO). 

It  is  better  to  use  the  reagent  when 'freshly  prepared.  At  all 
events  it  must  be  kept  in  the  dark  since,  as  remarked,  it  quickly 
decomposes  in  the  light.  It  serves  as  a  test  of  cellulose.20 


17.    MERCURIC  CHLORIDE.    Hg  C12. 
(Corrosive  sublimate.) 

It  may  be  had  in  the  market  sufficiently  pure  in  little  white 
sparkling  crystals  and  should  be  used  in  very  dilute  aqueous  or 
alcoholic  solutions,  for  example,  1  part  mercuric  chloride  and 
100  parts  water,  or  2  parts  of  the  sublimate  to  100  parts  of  al- 
cohol (Pfeffer).  Dippel21  used  a  still  more  dilute  solution  of 
the  sublimate  in  the  study  of  protoplasm,  1  part  sublimate  to 
500  parts  of  water. 


20  For  the  same  purpose  one  may  use,  even  if  less  advantageous,  a  solution  of  basic 
cupric  sulphate  in  ammonia,  also  of  basic  cupric  carbonate  in  ammonia,  finally,  also,  nickel 
oxide  ammonia.    (Schacht). 

21  Dippel,  Mikroskop,  Bd.  I,  p.  282. 


296  THE  MICROSCOPE  IN  BOTANY. 

18.     MiLLON'S  REAGENT     (Liqueur  nitromercurique.) 
Hg2  2  N  03  +  Hg  2  N  03  +  H  N  03. 


The  preparation  of  this  reagent  (quicksilver  and  quicksilver 
nitrate)  in  an  acid  solution  is  accomplished,  according  to  its 
discoverer  E.  Millon,  in  the  following  manner.22  Pour  upon  the 
pure  metal  an  equal  weight  of  nitric  acid  which  contains  4£ 
equivalents  of  water.  The  reaction  immediately  begins  in  a 
lively  way  in  the  cold.  When  it  begins  to  slacken  warm  gently 
till  the  metal  is  fully  dissolved.  But  directly  it  is  finished  add 
two  volumes  of  distilled  water  to  one  of  the  quicksilver  solution. 
After  some  hours  decant  the  fluid  part  which  stands  over  the 
crystalline  mixture  of  mercuric  and  mercurous  nitrate.  This 
fluid  reacts  cold  upon  albuminous  substances  —  its  reaction  is 
complete,  however,  only  at  60°  to  70°.  It  is  well  to  boil  the 
mixture  itself.  A  continual  contact  with  the  reage'nt  does  not 
change  the  red  matter.  It  should  be  remarked  that  neither  in 
the  mercuric  or  mercurous  nitrate,  nor  in  their  mixture  is  to  be 
sought  the  reagent  alone.  It  is  necessary  that,  to  a  solution  con- 
taining these  two  salts,  nitric  acid  should  be  added  before 
coloring  will  be  produced.  The  pure  mercuric  nitrate  which  is 
saturated  with  nitric  acid  reacts  similar  to,  but  not  so  well  as,  a 
saturated  nitric  acid  mixture  of  the  two  salts. 

According  to  Hartig23  the  mercuric  and  mercurous  nitrate 
can  be  prepared  by  dissolving  mercury  in  a  like  weight  of  foam- 
ing nitric  acid,  the  solution  afterwards  to  be  mixed  with  equal 
parts,  by  volume,  of  water.  The  reagent  should  be  used  only 
with  the  largest  cover-glasses  since  it  throws  off  acid  fumes. 
It  is  principally  a  test  for  albuminous  substances. 

19.    OSMIC  ACID    OsO4. 
(Perosnric  acid.) 

Osmic  acid,  or  perosmic  acid  as  it  is  commonly  named  in 
microscopy,  forms  colorless,  sparkling,  needle-like  crystals,  and 

*2  E.  Millon,  Sur  unreatif  propre  aux  composes  pratiques  (Annals  de  Chemie  et  de 
Physique,  Hie  Ser.,  tome  XXIX,  1850,  p.  507,  ff. 

23  Hartig,  I.  c.,  p.  154.—  Cfr.  also  Dippel,  I.  c.,  Bd.  I,  p.  281,  Poulsen,  1.  c.,  p.  29,  /.  (trans. 
p.  38).  Isageli  and  Schwendener,  I.  c.,  p.  475. 


ORGANIC   COMBINATIONS.  297 

should  be  used  in  a  very  weak  aqueous  solution.  The  solution 
of  this  expensive  reagent  (1  g.  costing  8  to  10  M.)  has  a  very 
disagreeable  smell,  like  chlorine,  and  the  vapor  from  it  attacks 
the  eyes  and  the  mucous  membrane  of  the  nose  in  a  very  un- 
pleasant way.  The  strength  of  the  solution  varies  between  1, 
and  0.1  per  cent.  Poulsen24  recommends  a  mixture  of  9  parts 
0.25  per  cent  chromic  acid  with  1  part  1  per  cent  osmic  acid 
for  the  preparation  of  young  meristem  tissue. 

Osmic  acid  has  a  manifold  application  as  a  microscopic  re- 
agent for  testing  fats  and  oils  and  for  the  study  of  protoplasm 
and  nuclei  (Strasburger),  and  for  hardening  cell  contents. 


B.     ORGANIC  COMBINATIONS. 

20.    ALCOHOL     (Ethyl  Alcohol)     C2H5.  HO. 

It  is  used  iu  the  anhydrous  state  (absolute  alcohol)  as  well  as 
diluted  in  various  degrees  with  water  and  with  glycerine  (p.  178) 
and  with  potassium  (p.  200).  It  can  be  purchased  sufficient- 
ly pure.  It  has  an  extended  usefulness  for  hardening  cell  walls 
and  cell  contents  (p.  178)  and  as  a  test  of  asparagiu,  iuuliu,  etc. 


21.     ETHER  (Ethyl  Ether)  (C2H5)2O. 

Commercial  ether  (sulphuric  ether)  has,  as  such,  an  abundant 
application,  for  drying  preparations  which  are  to  be  mounted  in 
Canada  balsam  (p.  222)  and  for  dissolving  resins,  fats,  and  es- 
sential oils  iii  plant  cells. 

22.    ACETIC  Aero  C2H4O2. 

(Glacial  acetic  acid.) 

The  concentrated,  or  the  so-called  "glacial"  acetic  acid  must 
be  considerably  reduced  before  it  can  be  successfully  used,  1 
vol.  acid  to  2,  3,  or  4  volumes  of  water.  Very  weak  (1  per 
cent)  aqueous  solutions  are  useful  in  the  study  of  nuclei  (Stras- 

24  Poulsen,  1.  c.,  translation  p.  20. 


298  THE  MICROSCOPE  IN  BOTANY. 

burger) .  Acetic  acid  was  also  applied  by  Hanstein  for  bleaching 
(see  p.  199),  for  tests  of  oxalic  and  carbonic  acid  salts  and  for 
making  nuclei  visible. 


23.     CUPRIC  ACETATE  Cu  (Q  H3  O2)2. 

Quite  pure  acetate  of  copper  can  be  found  in  the  market. 
It  may  be  purified  if  necessary  by  repeatedly  recrystallizing. 
The  saturated  aqueous25  solution  is  employed  in  microscopy  and 
is  used  as  a  test  of  turpentine  resin. 

24.    SODIUM  NITRO-PBTJSSIATE. 
Na2  Fe  C5  N5  (N  O)  +2  H2  O. 

We  use  the  commercial  crystallized  salt  which  must  be  kept 
in  an  air-tight  bottle.  It  should  be  prepared  fresh  on  the  rare 
occasions  when  we  shall  want  to  use  it  as  a  test  for  free  sulphur 
in  bacteria,  for  example. 

25.    POTASSIUM  FERROCYANIDE  K4  Fe  (C  N)6. 
(Yellow  prussiate  of  potash.) 

Will  be  used  in  rare  cases  in  an  aqueous  solution  as  a  test  for 
iron.  It  may  be  replaced  also  by  an  alcoholic  solution  of  pot- 
assium sulphocyanide  (CN.  S  K),  (Poulsen.) 

26.    OXALIC  ACID  C2H2O4. 

The  commercial  crystallized  acid  may  be  suitably  purified  by 
recrystallization.  In  its  aqueous  and  alcoholic  solutions  it  comes 
into  frequent  use  as  a  stain  for  sections  as  will  be  shown  later. 

COOH 
27.     ASPARAGIN  C2H3(NH2)< 

C  O.  N  H2 

Borodin26  used  a  concentrated  aqueous  solution  of  asparagin 

• 

25  Franchimont  in  Archives  Neerlandaises,  t.  VI,  1871,  p.  427. 

26  Borodia  in  Botan.  Zeitung,  1878,  p.  804. 


ORGANIC  COMBINATIONS.  299 

as  a  test  of  asparngin  crystals  in  etiolated  vegetable  tissue. 
According  to  Poulsen,27  asparagin  is  best  prepared  by  the  evap- 
oration of  the  boiled  and  filtered  sap  of  young  etiolated  legum- 
inous sprouts  (Lupinus),  or  by  evaporating  the  dialyzed 
aqueous  extract  of  althese  root.  The  asparagin  then  crystal- 
lizes out. 

28.    CANE  SUGAR  C^H^On. 

This  substance  may  be  properly  used  in  microscopical  analysis 
in  the  form  in  which  it  appears  in  the  pharmacopoeia  under  the 
name  of  Syrupus  simplex.  It  serves,  as  Kaspail  first  found,  as 
a  test  with  sulphuric  acid  for  proteids. 


29.    ANILINE  COLORING  MATTER. 

Recently  numerous  aniline  colors  have  been  recommended  for 
staining  sections  of  plant  tissue,  after  being  first  introduced  by 
Hanstein  as  histological  reagents  in  microscopical  botany.  Now 
a  large  number  of  aniline  colors  is  prepared  as  staining  media. 
For  example,  aniline  green  (methyl  green),  methyl  violet,  fuch- 
sin,  iodine  green,  aniline  brown,  bismark  brown,  saffranin, 
magdala,  gentian  violet  and  others.  We  shall  not,  however, 
give  all  the  proposed  methods  of  staining.  Many  of  them, 
especially  such  as  have  been  discovered  in  England  and  Amer- 
ica, are  not  suitable  for  a  scientific  work  but  offer  the  means 
only  of  a  very  pretty  pastime,  since  they  do  indeed  give  to  a 
section  under  examination  a  very  beautiful  color,  but  do  not 
differentiate  any  histological  relations.  Whoever  desires  recipes 
for  these  beautiful  colors  may  look  for  them  in  microscopical 
journals  written  in  English,  which  commonly  swarm  with  them. 
We  mention  here  only  : 

•1.  Hanstein 's  aniline  mixture?*  Hanstein's  method  of  ani- 
line staining  is  to  this  day  still  the  most  useful.  His  aniline 
mixture  is  prepared  in  the  following  manner.  Take  like  parts 

"  Poulsen,  /.  c.,  p.  34.  Trans.,  p.  45. 

28  Hanstein  in  Sitzuugsber.  d.  natur.  Ver.  der  pr.  Rheinland  u.  Westfalens,  1868.  —  Bot. 
Zeitung,  1808.  p.  708,  ff. 


300  THE  MICROSCOPE  IN  BOTANY. 

of  aniline  fuchsin  and  methyl  violet,29  pulverize  and  intimately 
mix,  till  they  form  a  cloudy  violet  powder.  Dissolve  as  much 
of  this  mixture  in  absolute  alcohol  as  will  produce  a  concentrated 
solution,  which  appears  in  the  glass  flask  where  it  is  kept  per- 
fectly black  with  a  purple  metallic  luster.  For  most  purposes 
this  concentrated  solution  is  used  direct.  For  many  highly 
colored  tissues  instead  of  this  a  solution  diluted  with  alcohol. 
The  degree  of  dilution  may  be  chosen  according  to  the  nature 
of  the  object  stained.  The  dilute  solution  should  contain  as 
much  aniline  as  one  can  see  through  without  difficulty  when 
put  in  a  test  tube  of  average  caliber. 

The  methods  of  staining  in  all  aniline  mixtures  are  essentially 
the  same.     They  will  now  be  briefly  pointed  out. 

(a)  Pour  a  few  drops  of  the  aniline  solution  into  a  watch- 
glass  and  transfer  the  section  from  absolute  alcohol  to  it  for  a 
few  moments,  as  many  as  necessary  to  produce  the  best  effect, 
holding   it  fast   with  the  forceps.     Then  dip  it  repeatedly  in 
absolute  alcohol  till  all  adhering  aniline  disappears  and  transfer 
it  to  the  slide  into  water  or  glycerine. 

(b)  Transfer  the  section  from  the  alcohol  to  the  slide  and 
with  blotting  paper  or  the  like,  remove  all  superfluous  alcohol, 
not  so,  however,  that  the  section  becomes  dry.     Now  give  it  a 
drop  of  the  aniline  solution   (nearly  or   quite   concentrated), 
and  let  the  section  so  far  dry,  that  it  is  only  just  damp.     Then 
flow  the  section  with  alcohol  till  all  adhering  aniline  is  dissolved 
and  washed  away,  letting  the  flowing  alcohol  run  off  the  oblique- 
ly  held   slide  into   a   vessel  prepared   for  it.     After  washing 
add  a  drop  of  water  or  glycerine  and  put  on  the  cover-glass  (or- 
ally communicated  to  me  by  Han  stein). 

2.  Aniline  fuchsin  solution.     It  is  prepared  by  dissolving 
aniline  fuchsin  in  absolute  alcohol,30  or  in  like  parts  of  water 
and  alcohol.81^  The  latter  should  be  used  to  test  corky  mem- 
branes. 

3.  JFi'e$*s  fuchsin  solution.32     It  consists  of  1  g.  crystallized 
fuchsin,  20  to  25  dropsv  absolute  alcohol  and  15  cc.  of  water. 


: 


29  Or  two  pai-ts  aniline  fuchsia  and  one  part  methyl  violet. 

80  Poulsen,  1.  c.,  p.  40,  trans.,  p.  59. 

si  Oliver  in  Bull,  de  la  Soc.  bot.  de  France,  t.  XXVII,  1880,  p.  234,  /. 

»2  Frey,  I.  c.,  p.  97. 


OEGANIC  COMBINATIONS.  301 

It-  may  be  applied  to  plant  tissue  for  comparison  with  the  effect 
produced  by  Hanstein's  aniline.  The  corresponding  blue  fluid 
is  prepared  by  dissolving  0.02  g.  aniline  blue  in  20  to  25  drops 
absolute  alcohol  and  25  cc.  of  water.33 

4.  Methyl  violet  of  Koch.**     A  few  drops  of  a  concentrated 
solution  of  methyl  violet  in  absolute  alcohol  added  to  20  cc.  of 
distilled  water,  thereby  producing  an  intensely  yellow  colored 
fluid.     This  fluid  is  used   for  coloring   bacteria    (see   below) 
and  after  the  staining  is  ended  the  preparation  should  be  washed 
with  water  or  a  dilute  solution  of  potassium  acetate.  .  For  the 
same  purpose  an  aniline  brown  is  recommended. 

5.  Methyl  green,  in  a  not  too   concentrated   solution  with 
(Strasburger),  or  without  (Treub)  1  per  cent  acetic  acid  colors 
nuclei  and  chlorophyll  grains  (Hanstein),  green. 

6.  Picro-anttine.9    Two  to  three  cc.  of  a  saturated  solution 
of  aniline  blue  (bleu  de  unit)  are  mixed  with  50  cc.  of  a  satur- 
ated solution  of  picric  acid  (especially  good  for  animal  tissues 
not  tested  by  me).36 

30.    ANILINE  SULPHATE    2  C6  H7  N.  S  O4  H2. 

This  substance  which  can  be  had  in  the  market  is  a  violet 
brown  powder.  It  is  used  in  a  concentrated  solution.37  It  is 
advantageous  to  acidulate  this  solution  somewhat  with  sulphuric 
acid.  It  may  be  kept  for  years  without  losing  its  virtue.  It 
colors  cell  walls  containing  liguin  gold-yellow. 
f  According  to  Wiesncr38  the  aniline  sulphate  of  the  market  is 
pure  enough,  while  von  Hohnel39  contradicts  this  and  uses  in- 
stead muriatic  aniline.  This  he  dissolves  in  water  and  strongly 

38  Frey,  1.  c.,  p.  98. 

»*  Koch  in  Cohu's  Beitr.  z.  Biologia  d.  Pfl.  Bd.  IT,  p.  400. 

35  Bachmann,  Leitf.  z.  Aut'ert  mikr.,  DanerprUparate,  MUnchen,  1879,  p.  28. 

96  Cf.,  also  \V.  Sterling  in  Journ.  of  Anat.  andPhysiol.,  Vol.  XV,  1881,  pp.  349-351,  which 
proposes  numerous  complicated  staining  media,  viz.,  picro-carmine  and  aniline,  picro- 
carmine  and  iodine  green,  eosin  and  iodine  green,  gold  chloride  and  aniline  (not  all  tested). 
Cf.  further.  Journal  of  the  Royal  Microscopical  Soc.,  Ser.  II,  Vol.  I,  p.  527,jf.,finally,  I.  c., 
p.  8(58,  where  Richardson  proposes  to  color  vegetable  tissue  with  Atlas-scharlach,  solution 
of  aniline  blue,  iodine  green,  and  malachite  green. 

17  Burgerstein  in  Sitzungsber.  d.  K.  K.  Akad.  d.  Wise.  Wien,  Bd.  LXX,  1  Abth.,  1S74,  p. 
341. 

38  Wiesner,  The  same  publication,  Bd.  LXXVII,  1  Abth.,  1878,  p.  60. 

89  v.  Hohnel,  The  same  publication,  Bd.  ULXVI,  1  Abth.,  1877,  p.  527. 


302  THE  MICROSCOPE  IN  BOTANY. 

acidulates  with  hydrochloric  acid.  The  alcoholic  muriatic  ani- 
line solution  with  the  subsequent  addition  of  water  is  also  re- 
commended. 


31.     PHENOL     (Carbolic  acid)  C6  H5.  O  H. 

This  acid  alone  is  not  often  used,  but  in  connection  with  hy- 
drochloric acid  (phenolhydrochloric  acid),  it  has  been  recently 
proposed  by  v.  Hohnel40  as  a  test  for  wood  substances. 

The  phenolhydrochloric  acid  of  v.  Hohnel  is  prepared  by 
making  a  concentrated  solution  of  crystallized  carbolic  acid  of 
the  utmost  possible  purity  in  concentrated  hydrochloric  acid. 
The  carbolic  acid  is  dissolved  in  the  least  possible  amount  of 
muriatic  acid  by  the  application  of  heat,  and  while  it  is  cooling 
as  much  more  muriatic  acid  is  slowly  added  as  will  clear  up  the 
existing  cloudiness. 

32.    PHLOKOQLTJCIN  (Trioxhydrobenzol)  C6  H3.  (O  H)3. 

This  substance  widely  distributed41  in  the  vegetable  kingdom 
is  in  a  pure  state  a  small,  bright  yellow,  transparent  crystal  solu- 
ble in  both  water  and  alcohol.  Phloroglucin  forms  at  the 
present  time  one  of  the  best  reagents  we  have  for  testing  ligni- 
fied  cell  walls.  The  following  sketch  of  the  discovery  of  the 
reagent  will  not  be  uninteresting. 

Y.  Hohnel42  having  put  a  section  of  a  twig  of  Salix  purpurea 
in  hydrochloric  acid  observed  that  the  woody  membranes  of  the 
section  was  colored  an  intense  -violet.  He  ascribed  the  col- 
oring to  a  substance  frequently  occurring  in  the  vegetable  king- 
dom and  which  he  called  xilophilin  and  whose  presence  he 
detected  in  143  species  of  plants.  Wiesner43  afterwards  more 
exactly  investigated  the  xilophilin  of  Hohnel  and  found  it  to  be 
a  mixture  of  phloroglucin  and  pyrochatechtn.  It  further  ap- 

40  v.  Hohnel,  1.  c.,  p.  528,  p.  700,  ff. 

41  Th.  v.Weinzierl.  Concerning  the  distribution  of  phloroglucin  in  the  vegetable  king- 
dom (Oesterr.  Bot.  Zeitschr.,  1876,  p.  285-304). 

42  F.  v.  Hohnel,  Histo-Chemical  investigations  of  xylophilin  and  coniferin.    Sitzungs- 
ber.  K.  K.  Akad.  Wein,  Bd.  LXXVI,  1  Abth.,  1877,  pp.  663-710. 

43  J.  Wiesner,  Note  concerning  the  action  of  phlorojducin  and  some  related  bodies  upon 
lignified  cell  membrane  (last  cited  publication,  Vol.  LXXV11, 1  Abth.,  Ib78,  pp.  60-66. 


ORGANIC  COMBINATIONS.  303 

peared  that  phloroglucin  was  a  most  sensitive  reagent  on  wood 
substances,  and  that  commonly  lignified  tissue  treated  to  hydro- 
chloric acid  served  as  a  most  sensitive  reagent  for  phlorogluciu. 

Phloroglucin  is  used  iu  an  aqueous  or  alcoholic  solution. 
Wiesner  recommended  a  9  per  cent,  but  still  gave  also  a  0.01, 
0.005,  even  0.001  per  cent  solution  as  capable  of  making  a 
reaction. 

Since  phloroglucin  is  a  very  expensive  article  at  this  time, 
and  not  to  be  had  by  every  one,4*  a  cherry-wood  extract  con- 
taining phloroglucin  may  be  used,  which  is  prepared  in  the  fol- 
lowing manner.45 

Not  too  small  branches  of  cherry  tree  are  washed  and  fastened 
into  a  bundle  and  then  by  means  of  a  plane  cut  into  fine  chips 
or  shavings.  Pour  alcohol  over  these  letting  it  stand  twenty- 
four  hours  to  extract  the  chlorophyll,  which  much  disturbs  the 
action  of  the  reagent,  and  for  this  reason  not  too  young  twigs 
should  be  used  on  account  of  their  having  so  much  chlorophyll. 
Turn  off  the  first  and  replace  with  a  fresh  quantity  of  alcohol, 
and  let  it  stand  for  several  days,  frequently  stirring.  Then  filter 
the  extract  and  evaporate  the  fluid  almost  entirely  away,  till  a 
piece  of  blotting  paper  which  has  considerable  wood  fibre  in  it> 
dipped  first  in  hydrochloric  acid  and  then  into  the  solution  turns 
rapidly  and  intensely  violet.  In  this  way  one  gets  a  brown 
liquid  which  smells  like  camphor. 

33.     INDOL  N  C8  H7. 

Xiggl46  has  recently  recommended  this  very  unpleasant  smell- 
ing stuff47  as  a  reagent  for  lignified  cell  membranes.  It  cannot 
be  used  in  an  alcoholic  solution  since  it  spoils  in  a  few  days.  It 
is  but  little  soluble  in  water  and  one  can  work  for  months  with 
a  few  little  crystal  plates  for  a  solution.  The  best  way  of  mak- 
ing the  solution  is  by  warming  the  water.  It  should  be  em- 

44  To  be  had  of  Dr.  Theodor  Schucharclt,  Gb'rlitz,  Schlesien  Germany.  Also  of  J.  T. 
Brown,  cor.  Washington  and  Bedford  Sts.,  Boston,  Mass.  Mr.  Browu  also  will  keep  on 
hand  all  the  micro-chemical  reagents. 

«  v.  Hohnel,  1.  c.,  p.  685. 

46  M.  Niggl.    Indol  a  reagent  on  lignified  cell  membranes.  Microchemical  investigations 
(Flora.  1881.  pp.  545-559,  pp.  561-5G6). 

47  Beyer  in  Ann.  Cheui.  Pharm.,  Bd.,  CXL,  p.  1,  ff,  p.  295,/. 


304  THE  MICROSCOPE  IN  BOTANY. 

ployed  with  sulphuric  acid  of  the  specific  gravity  of  1.2,  which 
consists  of  1  vol.  English  sulphuric  acid  diluted  with  4  vol. 
water  (cfr.  p.  236). 

34.    EOSIN  C2oH8B4O5. 

In  a  weak  aqueous  solution  this  reagent,  which  may  be  had 
free  from  arsenic  in  the  market,  is  used  according  to  Poulsen48, 
to  color  bacteria,  also  according  to  this  author  it  colors  dead 
protoplasm  a  rose-red  excellently  well.  It  has  recently  been 
recommended  for  double  staining  of  the  tissue  of  the  higher 
plants,  the  methods  of  which  require  further  testing.49 


35.    HJEMATOXYLIN    C16  H,4  O,  +  3  H2  O. 
(Extract  of  Logwood.) 

This  reagent  may  be  had  perfectly  pure  in  the  market.  Boh- 
mer  introduced  it  into  histology.  Frey60 gives  the  following  form- 
ula for  the  preparation  of  hsematoxylin  in  solution.  Dissolve  1  g. 
of  the  coloring  matter  in  absolute  alcohol.  Then  prepare  an 
alum  solution  of  0.5  to  1  g.  in  30  cc.  distilled  water.  Into  this 
drop  the  alcoholic  solution  till  it  has  attained  a  deep  violet  color. 
The  fluid  should  now  be  allowed  to  stand  some  days  in  the  air 
and  then  filtered ;  also,  afterwards  it  must  be  filtered  from  time 
to  time.  Duration  of  staining  process  from  5  to  30  minutes. 
Wash  with  distilled  water.  Over  colored  preparations  may  be 
bleached  by  putting  them  in  a  solution  of  alum. 

According  to  Poulsen51  0.35  g.  hsematoxylin  should  be  dis- 
solved in  10  g.  water  and  to  this  should  be  added  a  few  drops 
of  an  alum  solution  which  consists  of  3  g.  alum  and  30  g.  of 
water. 

It  may  be  added  that  hsematoxylin  colors  the  more  intensely 
the  more  alum  it  contains,  but  at  the  same  time  the  section  is 
made  more  brittle. 

48  Poulsen,  Om  nogle  mikroskopiske  Plantorganismer;   Nat.  Foren.  vidensk,  Medd' 
Kobenhavn,  1876-80,  p.  235  (separatabz,  p.  7).    Botanisk  Mikrokemi,  p.  89  (Trans,  p.  57). 
*»  Anicr.  Monthly  Microscop.  Journal  1880,  p.  81,  ff. 
eo  Frey,  1.  c.,  p.  99. 
ei  Poulsen,  L  c.,  p.  98,  translation,  p.  56. 


ORGANIC  COMBINATIONS.  305 

Poole52  makes  a  double  staining  of  vegetable  tissue  with  hae- 
matoxyliu  and  a  dilute  aniline  solution. 

For  animal  tissue  Frey,53  after  Stralzoff,  recommends  a  double 
staining  with  haematoxylin  and  an  ammoniacal  carmine  solution 
(see  p.  306),  which  method  may  be  applied,  probably  un- 
changed, to  vegetable  tissue.  The  section  should  be  stained 
with  haematoxylin  washed  with  distilled  water,  and  then  laid  in 
the  carmine  fluid ;  after  that  it  should  be  again  washed,  and 
finally  subjected  to  the  influence  of  a  weak  solution  of  alum. 
It  does  not  keep  well  in  glycerine.  According  to  Schmitz,5* 
preparations  hardened  in  picric  acid  are  especially  adapted  to 
staining  with  hsematoxylin.  HaBtnatoxylin  colors  nuclei  a  deep 
blue,55  and  may  be  applied  to  the  staining  of  bacteria.56 


36.    COCHINEAL  EXTRACT. 

An  aqueous  extract  of  the  pulverized  insect  prepared  with 
heat  contains  carmine  acid  (C17  H18  O10) ,  and  is  useful  in  staining 
vegetable  tissue.  It  is  very  liable  to  mould  and  so  must  be 
protected  by  a  few  drops  of  carbolic  acid.  Before  using,  a  few 
drops  of  acetic  acid  or  solution  of  alum  should  be  added. 

Czokor57  has  recently  recommended  a  cochineal  carmine  so- 
lution which  is  notable  for  being  capable  of  preservation  un- 
changed for  a  long  time.  Triturate  1  g.  cochineal  with  1  g. 
burnt  alum  to  a  fine  powder.  Then  add  100  cc.  of  distilled 
water  and  boil  till  it  is  but  60  cc.,  cool,  add  a  few  drops  of  car- 
bolic acid  and  filter  several  times.  The  resulting  solution  is  a 
beautiful  carmine  color  and  may  be  kept  without  change  for  six 
months.  Then  add  again  a  little  carbolic  acid  and  filter. 

The  cochineal  colors  bast  elements,  also  many  wood  cells, 
proteid  bodies  and  cell  nuclei. 

62  Poole,  in  Quart.  Jour,  of  Micros.  Science,  New  Series,  Vol.  XV,  1875.  p.  375,  ff. 
53  Frey,  I.  c..  p.  101.— Cf.  lurther  Brandt  in  Biolog.  Centralbl.,  1881,  p.  2O2.  ff. 
64  Sclimitz  in  Sitsungsber.  der  niederrh.  Gesellsch.,  Bonn,  1880,  Jahrg.  XXXVII,  p.  160. 
M  Johow,  The  cell  nuclei  of  the  higher  monocotyledons,  Bonn,  1880,  p.  9. 
66  Koch  in  (John's  Beitr.  z.  Biol.  d.  Pfl.,  Bd.  II.  p.  421.— Poulsen,  I.  c. 
57  Joh.  Czokor  in  Archiv  fur  mikrosk.,  Anatomic,  Bd.  XVIII,  1SSO,  p.  412.  jf. 
20 


306  THE  MICROSCOPE  IN  BOTANY. 

37.     CAKMINE  SOLUTIONS  (Carmine  red  CUH12O7). 

Commercial  carmine  is  the  coloring  matter  with  which  it  was 
first  attempted  (by  Th.  Hartig)  to  stain  anatomical  prepa- 
rations. Hartig  is  also  the  inventor  of  the  method  of  stain- 
ing as  mentioned  on  p.  267.  Since  the  introduction  of  this 
staining  medium,  many  naturalists  have  employed  various  mix- 
tures of  which  the  most  important  are  the  following. 

1.  Hartig 's  ammoniacal  carmine.5*      Commercial  carmine  is 
mixed  with  water  and  ammonia  fluid  is  added  in  drops  till  a 
perfect  solution  results.     The  solution  should  then  be  filtered, 
and  by  very  gentle  heat  evaporated  to  dryness.     This  carmine 
ammonia  thus  prepared  may  be  dissolved  in  water  and  can  be 
kept  for  years  in  good  condition  as  an  aqueous  solution. 

2.  Gerlach's  ammonium-carminate.59  According  to  Frey,60  this 
is  prepared  best  in  the  following  way.     Take  0.2  to  0.4  g.  of 
carmine,  mix  with  30   cc.   of  water  and  add  a  few  drops   of 
ammonia.     Thus  a  part  of  the  carmine  will  be  dissolved  and 
the  fluid   should  be  filtered.     The  rest  which  remains  behind 
may  be  kept  over  for  future  use.     If  the  filtrate  smells  at  all 
strongly  of  ammonia  it  should  be  permitted  to  evaporate  for  half 
or  a  whole  day  under  a  glass  bell.     If  after  the  lapse  of  time 
grains  of  carmine    begin  to  be  deposited  a  drop   of  ammonia 
will  restore  the  solution.     In    order  to  get  any  desired  color 
from  this  mass  it  should  be  transferred  to  water,  drop  by  drop, 
the  color  growing  of  course  from  a  light  to  a  darker  and  more 
intense  red. 

3.  Frey's  glycerine  carmine.*1    Dissolve  0.2  to  0.4  g.  of  car- 
mine in  the  required  amount  of  ammonia  and  add  30  cc.   of 
distilled  water.     To  the  filtered  fluid  add  30  g.  of  good  glycer- 
ine and  8  to  11  g.  of  strong  alcohol.     The  tincture   should  be 

O  o 

used  unmixed,  or  with  a  further  addition  of  glycerine. 

4.  TltierscJi's  oxalic  acid  carmine.6'2     One  g.  carmine  is  dis- 

68  M.  Hartig,  Entwick'ungsgesch.  d.  Pflanzenk.  p.  154.  —  Dippel,  I.  c.}  Bd.  I,  p.  284.— 
Poulsen,  1.  c.,  p.  3(>,  trans,  p. 49. 

6*  Mikrosk.  Studien  aus  d.  Gebiete  der  menschl.  Morphologic,  Erlangen,  1858. 

eo  Frey,  1.  c.,  p.  9:5. 

ei  Frey,  J.  c.,  p.  94. 

ea  Frey,  I.  c.,  p.  94.— Dippel,  I.  c.,  Bd.  I,  p.  285.— Poulaen,  I.  c.,  p.  36,  /.,  trans,  p.  50. 
Baclmianu,  I.  c.,  p.  62. 


ORGANIC  COMBINATIONS.  307 

solved  ill  1  cc.  of  ammonia,  and  mixed  with  3  cc.  of  distilled 
water.  Also  dissolve  8  g.  crystallized  oxalic  acid  in  175  cc.  of 
distilled  water.  Then  mix  the  solutions,  add  16  cc.  of  absolute 
alcohol  and  filter.  If  the  solution  has  an  orange  color  which 
comes  from  a  predominance  of  the  oxalic  acid,  it  can  be  cor- 
rected by  carefully  adding  drops  of  ammonia.  If  there  are  in 
addition  deposits  of  crystals  of  ammonia  oxalate  in  the  filtrate, 
which  may  happen  from  the  addition  of  the  ammonia  or  the 
alcohol,  the  fluid  must  be  filtered  the  second  time  ;  subsequent 
occasional  filtering  is  also  beneficial.  The  tincture  stains  very 
quickly.  The  coloring  matter  which  adheres  to  the  object  should 
be  washed  off  with  80  per  cent  alcohol.  If  the  color  becomes  too 
dark  or  diffuse,  soak  the  preparation  out  in  an  alcoholic  solution 
of  oxalic  acid. 

5.  Thiersch's  borax  carmine.™  Dissolve  2  g.  borax  in  28  cc. 
distilled  water  and  add  0.5  g.  pulverized  carmine.  To  the  red 
solution  thus  produced  add  60  cc.  of  absolute  alcohol  and  filter. 
(If  on  the  filter  paper  there  remains  a  mixture  of  undissolved 
carmine  and  borax  it  may  be  dissolved  in  distilled  water  and 
kept  over  for  future  use.)  For  soaking  out  use  an  alcoholic 
solution  of  oxalic  acid,  or  boracic  acid.  The  mixture  colors 
somewhat  slowly  but  very  beautifully.  According  to  Frey  (I.e.  ) 
one  gets  the  most  beautiful  coloring  when  one  lays  the  prep- 
aration for  a  moment  in  the  solution  after  it  has  been  previously 
impregnated  with  boracic  acid.  Studies  of  vegetable  nuclei 
are  very  essentially  facilitated,  according  to  Stras  burger,  by  the 
use  of  this  carmine  mixture,  the  color  making  the  form  of  the 
nucleus  come  out  most  beautifully.  The  preparations  should 
be  ex  <  mined  in  glycerine  and  mounted  in  that  or  in  glycerine 


6.  BeaVs  carmine  solution.^  Put  0.6  g.  of  pulverized  car- 
mine in  a  test  tube,  pour  over  it  2.3  cc.  of  concentrated  am- 
monia fluid  and  heat.  After  the  solution  is  completed  let  it 
stand  for  an  hour  and  pour  the  red  fluid  into  a  mixture  which 
is  made  of  60  cc.  of  water,  47.5  cc.  of  concentrated  glycerine, 

•»  Frey,  1.  c.,  p.  64.  —  Dippel,  I.  c.,  Bd.  I,  p.  285.  —  Strasburger,  Zellbild.  u.  Zelltheilung, 
1880,  p.  9. 

64  Frey,  I.  c.,  p.  95.    Poulsen,  L  c.,  p.  37,  trans,  p.  52. 


308  THE  MICROSCOPE  IN  BOTANY. 

and  19   cc.  of  absolute  alcohol,  stir  up  with  a  glass  rod,  let  it 
stand  for  some  time  and  then  filter. 

For  the  study  of  the  protoplasmic  contents  of  the  cells  in 
filamentous  algae  (Spirogyra)  one  should,  according  to  Stras- 
burger,65  lay  the  fronds  in  a  1  per  cent  solution  of  chromic  acid, 
for  at  least  four  hours.  Then  repeatedly  wash  in  distilled  water 
and  lay  in  a  mixture  of  Beal's  carmine  and  camphor,  diluted 
with  8  parts  water,  1  part  glycerine  and  1  part  alcohol.  There 
will  follow  after  some  time  a  rosy  coloring  of  the  protoplasmic 
cell  contents  which  makes  its  fine  structural  relations  very 
distinct. 

7.  Grenadier's  alum-carmine.*®    The  alum-carmine  represents 
an  exceptionally  fine  staining  fluid  for  cell  nuclei,  which,  accord- 
ing to  Grenadier  is   prepared  in  the  following  way.     Dissolve 
in  100  cc.  of  distilled  water,  0.5  to  1  g.  pulverized  carmine 
and  1    to    5  g.    potassium    alum    or   common   alum.     Tanglfi7 
recommends  a  like  composition  which  colors  nuclei  as  well  as 
cellulose  membrane.     "Dissolve  alum   in  water  to  saturation, 
mix  the  solution  now  with  any  desired  quantity  of  carmine,  boil 
about  ten  minutes  and  filter  after  cooling."     The  staining  re- 
quires from  5  to  10  minutes.      The  preparation  keeps  very  Avell 
in  glycerine,  and  the  use  of  this  stain  gives  very  instructive 
specimens.     In  order  to  obtain  clean  specimens  it  is  very  much 
recommended  to  previously  harden  the  part  of  the  plant  which 
is  to  be  stained  in  absolute  alcohol.     This  not  only  facilitates 
the  imbibition  of  the  stain  but  also  has  the  further  advantage 
that  the  staining  capacity  of  the  substances  in  the  cells  remains 
unchanged. 

8 .  The  Schweigger-Seidel  acid  carmine  volution ,68  A  common 
ammoniacal  carmine  solution  is  mixed  with  an  excess  of  acetic 
acid  and  filtered.     This  tincture  stains  diffusely  at  first.     Then 
put   the  colored   preparation  in   glycerine  to  which   has  been 
added  ^<hr  part  of  muriatic  acid.     This  will  remove  the  color 
from  the  cell  body  generally  leaving  the  stain  only  in  the  nu- 

e5  Strasburger.  1.  c.,  p.  172. 

Grenadier  in  Archiv  fiirMikrosk.  Anatomie,  Jahrg.  1879,  p.  4(>5,  thence  in  Zeitschrift 
f.,  Mikroskopie,  Jahrg.  II,  1879,  p.  55. 

67  E.Tangl  in  Pringsheims'  Jahrb.,  Bd.  XII,  1880,  p.  170. 

68  Frey.f,  c.,  p.  96. 


ORGANIC  COMBINATIONS.  309 

cleus.     In  order  to  mount  in  glycerine  one  should  wash  the 
preparation  in  water  containing  acetic  acid. 

All  carmine  mixtures  are  used  principally  for  staining  nitro- 
genous, most  of  all  protoplasmic,  substances.  The  nucleus 
takes  the  stain  with  particular  avidity,  and  mostly  in  greater 
quantity  than  the  surrounding  protoplasm.  All  these  substances 
absorb  carmine  but  never  till  after  death.  Cellulose  (with  the 
exception  of  ^some  modifications),  starch  and  other  cell  elements 
without  nitrogenous  contents,  do  not  absorb  the  coloring  mat- 
ter of  most  of  the  carmine  compounds. 

38.       PlCRO-CAHMTKTATE    OF    AMMONIA. 

(Picro-Carmine.) 

A  rapidly  staining  medium  is  recommended  by  Treub  for  the 
investigation  of  nuclei  and  by  Weigert  for  studying  bacteria, 
and  which  was  first  introduced  into  zoo-microscopy  by  Ran- 
vier,  viz.  picro-carminate  of  ammonia.  According  to  Frey69  it 
is  thus  made.  To  a  concentrated  aqueous  solution  of  picric 
acid  add  to  saturation,  drop  by  drop,  an  ammoniacal  carmine 
solution.  Then  evaporate  to  one-fifth  the  original  volume.  The 
cold  solution  deposits  a  small  sediment  of  carmine.  Then  filter 
and  evaporate  to  dryness  when  a  red,  ochre-yellow  powder  will 
be  obtained.  Dissolve  portions  of  this  in  the  preparation  of  the 
reagent  1  g.  in  100  cc.  water.  Filtering  from  time  to  time  is 
indispensable. 

Baber  (1.  c.)  mixes  1  g.  of  carmine  in  4  cc.  of  concentrated 
ammonia,  and  200  cc.  of  water,  then  adds  5  g.  picric  acid  and 
shakes  up  and  decants  so  that  the  undissolved  excess  of  the 
picric  acid  remains  behind.  After  the  red  liquid  has  stood  for 
several  days  with  frequent  shaking,  it  is  put  in  a  shallow  dish 
in  the  air  to  dry.  The  red  powder  should  then  be  dissolved; 
2  parts  to  100  of  water,  and  after  some  days  filtered  through 
two  layers  of  paper.  The  fluid  should  now  be  yellowish  red 
without  smell  of  ammonia.  A  drop  on  white  filter  paper  gives 
on  drying  a  yellow,  red-bordered  fleck.  A  couple  of  drops  of 
carbolic  acid  keeps  the  stain  from  decomposition. 

6»  Frey,  1.  c.,  p.  96. 


310  THE   MICROSCOPE  IN  BOTANY. 

Weigert's70  formula  is  as  follows.  Pour  4  g.  common  am- 
monia over  2  g.  of  carmine  and  let  it  stand  for  twenty-four 
hours ;  now  all  that  is  soluble  is  dissolved.  Then  add  the  small- 
est possible  quantity  of  acetic  acid  till  the  first  faint  traces  of 
precipitation  are  seen.  After  standing  another  twenty-four 
hours  add  some  ammonia,  drop  by  drop.  According  to  Baber 
the  mounting  fluid  which  is  best  for  specimens  stained  with  this 
reagent  is  one  made  with  10  drops  glycerine,  10  drops  water, 
and  1  drop  of  the  reagent  itself.71 


39.    ALCANNA   TINCTURE. 

This  reagent  is  recommended  by  N.  J.  C.  Miiller  in  an  aque- 
ous alcoholic  solution  as  a  test  for  resins  and  essential  oils.  A 
preparation  of  sufficient  thinness,  from  some  part  of  the  plant 
that  has  little  drops  of  resin  in  the  cells,  should  be  put  on  the 
slide  and  with  it  a  clean  fragment  of  alcanna,  and  to  both  add 
a  drop  of  dilute  alcohol.  In  a  few  minutes,  two  or  three,  the 
resin  drops  in  the  cell  will  be  stained  a  lively  red,  and  strangely 
enough,  more  intensely  stained  than  the  surrounding  aqueous- 
alcoholic  pigment  solution.  Protoplasmic  masses  without  the 
resinous  substances  require  from  one-quarter  to  one-half  hour 
in  a  like  concentrated  pigment  solution  before  a  distinct  color 
is  perceived.  If  the  stained  section  is  treated  with  alcohol 
and  the  colored  drop  disappears,  no  more  treatment  of  the 
samo  drop  with  the  same  reagent  will  make  the  color  perceptible 
to  him.  The  following  is  the  best  process  for  preparations  in 
water.  Break  from  the  alcanna  a  thin,  even  scale  of  perhaps 
about  the  size  of  the  section  to  be  tested.  Rub  it  between  the 
clean  fingers  with  a  drop  of  water  to  remove  all  attached  pow- 
dered parts,  and  lay  it  on  the  section  which  is  covered  already 
with  water.  Then  put  011  the  cover-glass  and  at  the  edge  of 
this  a  drop  of  alcohol.  After  two  or  three  minutes  remove  the 

70  C.  Weigert,  Zur  Tecknik  rter  Mikroskop,  Bacteriemintersuchung.     Virchow's  Archiv 
f.  pathol.  Anatomie,  Bd.  LXXXVIII,  Heft  2,  8,  Folge,  Bd.  II,  Kelt  2,  1881,  pp.  275-315. 

71  Double  stainings  with  this  reagent  and  carmine,  aniline,  osmic  acid,  and  picric  acid 
have  been  frequently  prepared.    (Cf.  Jour.  Anat.  and  Phys.,  Vol.  XV,  1881,  pp.  349-351 
Jour.  Roy.  Microscop.  Society,  1881,  p.  528,  etc.) 


ORGANIC  COMBINATIONS.  311 

fragment  of  alcanna,  and  with  sufficient  magnification  you  will 
find  the  drops  in  question  stained  a  beautiful  red.  This  will  all 
happen  as  indicated  if  the  alcanna  is  rich  in  coloring  matter, 
but  a  very  poor  article  is  now  often  sold  in  the  market.  It 
is  self-evident  that  if  we  apply  the  above  mentioned  test  of 
washing  out  the  stained  section  with  alcohol  we  may  easily 
distinguish  between  the  drops  of  resin  and  essential  oils,  and 
those  of  fatty  oils.72 

72  N.  J.  C.  Miiller,  Untersnchung  iiber  die  Vertheilungder  Harze,  etc.,  in  Pflanzenkorper 
(Pringsheim's  Jahrb.,  Bd.  V,  pp.  387-421). 


312  THE  MICROSCOPE  IN  BOTANY. 


CHAPTER    Y. 

MICROSCOPICAL  INVESTIGATION  OF 
VEGETABLE  SUBSTANCES. 

FOR  such  a  complete  presentation  of  the  microscopical  inves- 
tigation of  the  elementary  substances  of  plants  as  is  possible 
fiom  the  present  standpoint  of  scientific  microscopy,  we  must 
first  of  all  begin  by  making  such  a  classification  of  the  very  elab- 
orate materials  at  hand  as  may  be  practically  and  scientifically 
justified.  Since,  as  we  have  already  remarked,  a  comprehensive 
presentation  of  this  matter  does  not  at  the  present  time  exist, 
we  must  here  build  from  the  very  foundation.  As  we  survey 
the  rich  amplitude  of  material  which  the  investigations  of  the 
vegetable  histologist  and  physiologist  as  well  as  those  of  the 
chemist  have  provided  for  us,  it  is  not  difficult  to  see,  that 
the  manifold  substances  which  compose  the  organs  of  plants 
may  be  divided  according  to  the  frequency  of  their  occurrence 
into  two  groups.  First,  we  recognize  a  large  series  of  plant 
substances  which  have  a  very  wide  distribution  in  the  vegetable 
kingdom.  We  need  refer  here  only  to  albuminous  bodies  which 
no  plant  lacks,  or  to  cellulose,  which,  except  in  the  very  lowest 
plants,  occurs  likewise  in  all.  So  also  starch,  plant  mucilage, 
sugars,  chlorophyll  combinations,  etc.,  are  widely  distributed 
throughout  the  plant  kingdom,  and  the  instances  in  which  they 
fail  are  comparatively  very  few.  In  contradistinction  to  these 
widely  distributed  bodies  stands  a  series  of  substances  whose 
occurrence  is  either  limited  to  small  groups  of  plants,  or  which  are 
produced  only  by  certain  growths, —  substances,  at  all  events, 
which  are  of  but  secondary  importance  in  the  building  up  of 
plants.  Belonging  to  this  group,  to  mention  some  bodies  of 
which  microscopical  analysis  has  already  taken  possession,  are 
the  coloring  matter  of  algae,  tannic  acid,  resin,  balsam,  terpene, 
essential  oils,  coniferiii,  chrysophanic  acid,  and  many  others. 


SUBSTANCES  OF  UNIVERSAL  DISTRIBUTION.  313 

It  is  also  not  to  be  forgotten  that  many  substances  which  are 
placed  in  the  latter  category  have,  perhaps,  a  much  wider  and 
more  general  distribution  in  nature  than  the  present  state  of 
our  science  would  justify  us  in  assuming.  Thus,  for  example, 
some  physiologists  have  supposed  that  coniferin  occurs  in  all 
woody  growths.  So  also  the  investigations  of  Weinzierl  (see 
p.  302)  have  shown  that  phloroglucin  which  hitherto  had  only 
been  prepared  synthetically  by  the  chemist  (benzole,  on  which 
for  the  3  atoms  of  hydrogen  are  founded  3  groups  of  hydrox- 
ides of  equal  value,  trioxhydro-benzol),  or  the  so-called  xylo- 
philin  of  v.  Hohnel  (see  p.  302),  which,  according  to  Weisner's 
investigations  represents  a  mixture  of  phloroglucin  and  pyro- 
catechin,  constantly  occurs  not  only  in  woody  but  also  in  many 
herbaceous  plants.  But  perhaps  as  a  parallel  to  such  statements, 
the  objection  may  be  raised  that  such  a  presentation  as  the  one 
in  hand  must  adapt  itself  exactly  to  the  present  state  of  our 
knowledge  of  these  matters,  and  thence  can  be  useful  but  for 
a  limited  period  of  time,  and  must  necessarily  become  unser- 
viceable with  the  further  progress  of  science. 

TTe*  now  first  consider  those  plant  substances  which  are  of 
universal  distribution  and  of  such  we  present  the  following. 
1,  Cellulose  and  its  modifications;  2,  Starch;  3,  Dextrine;  4, 
Vegetable  mucilage ;  5,  Gums;  6,  Inuliii ;  7,  Grape  sugar;  8, 
Cane  sugar;  9,  Albuminous  substances;  10,  Chlorophyll;  11, 
The  coloring  matter  of  flowers;  12,  Asparngin ;  and  13,  Inor- 
ganic vegetable  elements. 

The  question  now  lies  before  us,  according  to  what  points  of 
view  we  might  arrange  these  substances  into  a  continuous  series, 
and  according  to  which  of  these  points  of  view  we  should  arrange 
them  so  as  to  be  both  practically  and  scientifically  justified. 
Three  points  of  view  offer  themselves  to  us.  These  substances 
maybe  grouped' according  to  their  morphological,  physiological 
or  chemical  characteristics.  In  the  first  case  we  should  have  to 
place  those  things  together  which  have  a  like  or  similar 'ap- 
pearance :  thus,  in  the  first  place,  the  materials  forming  the  cell 
Avail,  then  the  solid,  and  finally  the  fluid  and  semi-fluid  cell 
contents.  ^Ye  should  then  also  distribute  into  one  and  the 
same  group,  starch  and  proteid  grains,  chlorophyll,  calcium 


314  THE  MICROSCOPE  IN  BOTANY. 

crystals,  etc.,  joining  things  which  are  altogether  different.  A 
classification  of  vegetable  substances  in  accordance  with  their 
physiological  functions  is  impracticable,  simply  on  the  ground 
that  we  know  very  little  of  what  part  many  substances  take  in 
the  vital  processes,  and  of  many  others  we  know  nothing  at  all. 
There  remains  to  us,  therefore,  only  the  classification  of  these 
substances  according  to  their  chemical  nature.  And  this  is  also, 
in  fact,  the  most  suitable,  and  the  following  chapter  makes  it  its 
principal  purpose  to  furnish  directions  for  determining  the  chem- 
ical nature  of  the  substances  occurring  in  the  interior  of  plants. 

We  make  the  classification  upon  the  inorganic  elements,  and 
the  absence  or  presence  of  nitrogen  gives  us  the  first  character- 
istic for  a  wide  classification.  The  first  division,  combinations 
containing  no  nitrogen,  we  designate  collectively  carbo-hydrates, 
and  the  second  division  nitrogenous  combinations.  The  car- 
bo-hydrates include  the  cellulose  and  the  rest  of  those  isomeric 
vegetable  substances  in  which  the  formula  C6H10O5  occurs.  To 
the  carbo-hydrates  also  belong  the  different  kinds  of  grape  sugars, 
C6  H12  O6,  and  the  cane  sugars,  C12  H22  On.  The  nitrogenous 
combinations  include  the  well-known  albuminous  substances 
(proteid  bodies),  vegetable  coloring  matter,  of  which  chloro- 
phyll may  be  designated  as  the  most  important,  and  finally  as- 
paragin  as  an  amido-combination. 

According  to  the  point  of  view  which  we  have  here  developed 
we  get  the  following  arrangement  of  those  vegetable  elements 
which  have  the  widest  distribution. 

CARBO-HYDRATES. 

Cellulose  Group  C6H10O3  Sugar  Group. 

1.  Cellulose.    4.  Vegetable  mucilage.  7.  Grape  sugar  C6  H12  O6 

2.  Starch.        5.  Gums.  8.  Cane  sugar    C^U^O^ 

3.  Dextrine.    6.  Inulin. 

NITROGENOUS  COMBINATIONS. 

9.  Albuminoids  (proteid  bodies). 

10.  Chlorophyll.  > 

11.  Coloring  matter  of  flowers     |  Vegetable  coloring  matter. 

12.  Amido  combinations,  asparagin. 

13.  Inorganic  vegetable  elements. 


CELLULOSE  AND  ITS  MODIFICATIONS.  315 

It  should  be  remarked  in  reference  to  this  arrangement  that 
the  substances  designated  w  matter  "  (Stoffe)  are  by  no  means 
such  in  the  chemist's  sense.  A  chlorophyll  grain,  for  example, 
consists  as  is  known  of  a  large  number  of  bodies  which  on 
their  part  again  are  not  to  be  considered  as  simple  chemical 
combinations.  We  should  regard  the  present  arrangement  as 
nothing  more  than  a  grouping  of  important  physiological  in- 
dividuals. 

In  the  following  analysis  we  shall  first  make  ourselves  ac- 
quainted in  general  with  the  qualities  of  each  of  the  substances, 
and  then  describe  the  methods  of  microscopical  reaction  to  be 
employed  with  each  in  order  to  recognize  it  with  certainty. 
Very  unimportant  reactions,  or  those  whose  value  has  not  yet 
been  sufficiently  established,  will  either  not  be  mentioned,  or 
will  be  but  incidentally  referred  to,  and  on  this  account  we 
once  for  all  refer  the  reader  to  the  "  literature  "  to  be  found  be- 
fore each  section. 


A.     SUBSTANCES    OF     UNIVERSAL    DISTRIBUTION. 

I.       CELLULOSE    AND   ITS   MODIFICATIONS. 

Cellulose  or  cell-substance  (C6  H10  O5  or  C]2  H^  O10)  presents 
in  its  pure  state  a  solid,  colorless  transparent  body.  Cellulose 
appears  in  plants  in  the  form  of  cell  cuticle  or  cell  wall.  It  is 
isomeric  to  the  other  members  of  the  group  of  cell  substances 
enumerated  on  p.  314,  but  in  part  varies  much  from  them 
in  its  chemical  and  physical  behavior.  The  isomerisin  explains 
the  fact  that  many  of  these  substances  during  the  vital  pro- 
cesses in  the  body  of  the  plant  can  be  easily  changed  into  one 
another,  as,  for  example,  a  transformation  of  starch  into  cellu- 
lose and  of  cellulose  into  gum  often  takes  place. 

Under  the  physiological  conception  of  cellulose  we  are  how- 
ever to  understand,  in  accordance  with  the  science  of  to-day, 
not  only  pure  cellulose,  but  also  certain  related  substances, 
which  result  from  the  metamorphosis  which  cellulose  undergoes 
in  the  life  processes  of  the  plant,  and  which  in  general  are  dis- 


316  THE  MICROSCOPE  IN  BOTANY. 

tinguished  by  containing  more  carbon  and  less  oxygen  than 
cellulose  in  the  strict  sense.  These  modifications  some  nat- 
uralists regard  as  different  chemical  individuals  (Fremy),  while 
the  majority  of  chemists  and  physiologists  see  in  them,  as  already 
mentioned,  only  modifications  (Payen,  Fromberg,  Mulder). 
Thus  Fremy  specifies  as  chemical  cellulose  individuals  of  this 
sort,  essential  cellulose,  paracellulose,  vasculose,  fiberose  and 
cutose.1*  If  we  can  at  present  form  no  definite  idea  of  the 
nature  of  the  modifications  of  cellulose,  it  still  appears  prob- 
able, in  opposition  to  the  view  of  Fremy,  and  in  accordance 
with  the  chemical  investigations  of  Payen  and  later  of  Schultze,2 
as  well  also  as  in  accordance  with  the  studies  of  the  botanists, 
that  the  change  in  the  cellulose  is  produced  by  the  molecular 
intercalation  during  the  process  of  growth,  of  certain  other 
substances  into  the  cell-wall  which  originally  consisted  of  pure 
cellulose.  For  example,  we  have  the  phenomenon  of  lignifica- 
tion,  whereby  the  cellulose,  as  is  well  known,  is  transformed 
into  lignin.  This  takes  place  by  the  intercalation  of  a  sub- 
stance, which  Payen  had  already  recognized  and  designated  by 
the  name  of  "  incrusting  substance,"  and  which  Schultze  after- 
wards believed  himself  to  have  prepared  pure. 

The  modifications  of  cellulose  which  have  been  distinguished 
with  sufficient  distinctness,  are  wood  substances  (lignin)  ; 
middle  lamella,  intercellular  substance,  which  very  much  re- 
sembles wood  in  many  respects;  cork  substance  (suberin)  out 
of  which  is  also  composed  the  corky  layer  known  as  cuticle 
which  regularly  covers  the  epidermis  (cutin),  and  the  inclosing 
layer  of  the  pollen  grain  (pollenin)  ;  finally,  fungus  cellulose. 
The  latter  to  which  one  might  suitably  give  the  name  of  fun  gin, 
if  this  had  not  previously  been  used  in  another  sense,  was,  till 
a  short  time  ago  looked  upon  by  botanists  of  repute3  as  an 

1  Fremy,  Comptes  rend.,  t.  XLVIII,  p.  667,  ff.,  p.  862,  ff. 

*  Fremy  at  present  classifies  the  constituents  of  vegetable  tissues  under  the  following 
seven  heads,  the  characters  being  derived  from  their  chemical  constitution.  1,  Cellulose 
substances  (cellulose,  paracellulose,  and  nietacellulose);  2,  Vasculose;  3,  Cutose;  4,  Pec- 
tose;  5,  Calcium  pectate;  6,  Nitrogenous  substances;  7,  Mineral  elements  (see  at  large, 
Ann.  Sci.  Nat.  XEII.1882,  pp.  360-382,  condensed  in  Jour.  Roy.  Micro.  Soc.,  Vol.  HI,  18*53,  pp. 
232-5).  A.  JB.  H. 

2  Schultze  in  Chem.Centralbl.,  1857,  p.  321. 

8  De  Bary,  Morphologic  d.  Pilze,  Flechten  u.  Myxomyceten  (Bd.  II,  von  Hofmeister's 
Handb.,  p.7,2F.). 


CELLULOSE  AND  ITS  MODIFICATIONS.  317 

isomeric  substance  to  cellulose  in  the  sense  of  Fiemy.  But 
according  to  the  latest  investigations  of  Richter,4  it  appears 
that  this  also  is  nothing  else  than  common  cellulose  with  foreign 
admixtures,  mainly  albuminous  substances.  On  the  contrary, 
it  remains  questionable  if  the  medulin  of  the  chemist  forms  a 
like  sharply  pronounced  modification  of  cellulose.  Omitting 
now  these  modifications,  we  have  still  to  describe,  under  the 
true  cellulose,  those  conditions  which  arise  from  the  disorgani- 
zation of  cellulose  and  lead  to  isomeric  combinations,  as  amy- 
loid, plant  mucilage,  caoutchouc,  arabin,  bassorin,  etc.,  and 
which  we  may  suitably  designate  by  the  expression  rnuculent  cel- 
lulose. 

Classified  according  to  their  characteristic  qualities,  cellulose 
and  its  related  substances  may  be  arranged  in  the  following 
manner : 

1.  Essential.  Cellulose,  cell  substance,  soluble  in  cupram- 
monia,    concentrated    sulphuric    and    chromic  acid.     It   colors 
blue  or  violet  with  iodine  and  sulphuric  acid,  or  with  chlor- 
iodide   of  zinc.      It   has  no  admixture  of  foreign  substances. 

2.  ^Incident    Cellulose,  frequently  soluble  in  cupraminonia 
as  well  as  in  concentrated  sulphuric   acid  and  chromic   acid. 
It  seldom  colors  blue  with  iodine  and  sulphuric  acid,  or  with 
chlor-iodide  of  zinc,  but  mostly  yellow  or  yellowish,  or  remains 
quite    colorless.      It  is  distinguished   from    all  other  forms  of 
cellulose  by  its  swelling. 

3.  Wood    Cellulose,   Lignin,    insoluble    in    cuprammonia, 
soluble  in  concentrated   sulphuric    and   chromic    acid ;    is  col- 
ored almost  always  yellow  with  iodine  and  sulphuric  acid,  or 
with  chlor-iodide  of  zinc.     With  phloroglucin  and  hydrochloric 
acid  rose  red  (distinguished  from  all  other  kinds  of  cellulose)  ; 
possesses  less  oxygen  than  pure  cellulose. 

4.  Middle   Lamella,   Intercellular  Substance,  insoluble    in 
cuprammonia,  insoluble  in  concentrated  sulphuric  and  chromic 
acid,  colors  yellow  with  iodine  and  sulphuric  acid,  or  with  chlor- 
iodide  of  zinc. 

«  Richter  in  Sitsungsber.K.  K  .  Acad.  d.  Wiss.  Wien,  Bd.  LXXXIII,  I  AUh.,  1881,  p.  510. 


318  THE  MICROSCOPE  IN  BOTANY. 

5.  Cork  Cellulose,  Suberin  (including  cutiu,  po'llenin),  in- 
soluble  in  cuprammonia,  insoluble   in   concentrated  sulphuric 
acid  and  chromic   acid  (or  in  the  latter  very  slightly  soluble), 
colors  very  seldom  yellow,  mostly  brown  with  iodine  and  sul- 
phuric acid  ;  gives  cerinic  acid  reaction  with  Schultze's  mixture. 
It  contains  an  admixture  of  certain  nitrogenous  substances. 

6.  Fungus    Cellulose,    insoluble    in    cuprammonia,    very 
slightly  soluble  in  concentrated  sulphuric  acid.     It  very  sel- 
dom takes  a   blue  color   with  iodine  and  sulphuric   acid,    or 
with   chlor-iodicle   of  zinc.      It   occurs  only   in   fungus    (and 
lichens)  and  appears  to  contain  an  admixture  of  albuminous 
substances. 

From  these  varieties  of  modified  cellulose,  may  be  obtained 
the  pure  cellulose  which  will  give  a  true  cellulose  reaction  with 
iodine  and  sulphuric  acid,  or  chlor-iodide  of  zinc,  if  they,  ac- 
cording to  their  nature,  be  treated  with  water,  or  with  alcohol 
or  ether,  dilute  acids,  nitric  acid,  together  with  potassium  chlo- 
rate, or  caustic  potash. 


1.    CELLULOSE  IN  THE  NARROW  SENSE.    (Cell  substance.) 


The  most  important  reagents  for  testing  cellulose  are  those 
iodine  solutions  particularly  described  on  p.  285  ;  in  contra- 
distinction to  these  the  other  methods  for  testing  cellulose  are 
of  a  very  subordinate  nature.  They  are  commonly  employed 
only  when  by  the  reactions  of  the  iodine  solutions,  we  are  not 
able  to  determine  with  definiteness  whether  we  have  pure  cel- 
lulose before  us  or  one  of  the  more  closely  related  modi- 
fications of  it.  In  this  case  one  must  observe  its  behavior  when 
treated  with  mineral  acids,  alkalies,  cuprammonia,  alum  car- 
mine, copper  sulphate  and  potassium,  or  in  a  secondary  way 
by  its  negative  behavior  towards  phenol-hydrochloric  acid,  ani- 
line sulphate,  phloroglucin  and  indol,  which  will  demonstrate 
its  distinction  from  other  cellulose  modifications. 


CELLULOSE  AND  IODINE  REAGENTS.  319 

A.     Behavior  of  Cellulose  to  Iodine  reagents. 

Literature.  J.  B.  Eead,  On  the  chemical  composition  of 
vegetable  membrane  and  fiber  (Lond.  and  Edinburgh,  Phil. 
Magazine,  Vol.  XI,  1837,  p.  421,  f.)  Schleiden,  Einige 
Bemerk.  iiber  die  sogen.  Holzfaser  der  Chemiker  (Wiegmann's 
Archiv,  Jahrg.,  IV,  1838,  Bd.  I,  p.  49, /I)  —  Schleiden,  Einige 
Bemerk.  iiber  d.  vegetabil.  Faserstoft'  und  sein  Verhalten  z. 
Starkmehl  (Poggendorff's  Annalen,  Bd.  XLIII,  1838,  p.  391). 
—Schleiden,  Beitrage,  etc.,  13,  160,  164,  172,  u.  a.  and  O. 
— Schleiden,  Noch  einige  Bemerk.  iiber  d.  veget.  Faserstoff, 
u.  sein  Verb.  z.  Starkmehl  (Flora,  1840,  Bd.  II,  p.  737,  ff., 
p.  753,  ff.) — Mohl,  Einige  Beobacht.  iiber  d.  blaue  Farbung 
d.  veget.  Zellmembran  durch  Jod  (Flora,  1840,  Bd.  II,  p. 
609,  f.,  p.  625,  ff.) — Payen,  Mem.  Stir  la  Compos,  chim.  du 
tissu  propre  des  veget.  phanerog.  (Ann.  des  sc.  nat.  2e  ser., 
t.  XIV,  1840,  pp.  73-100.) — Lantzius-Beuinga,  De  evol. 
sporid.  muse.  Gott.,  1840,  p.  7.— Mohl,  Verm.  Schriften.  Tub- 
ing., 1845,  p.  337,/*.,  u.  daselbst  a.  v.  a.  O. — Mohl,  Bildet  d. 
Cellulose  d.  Grundlage  sammtl.  veget.  Membraneii?  (Bot. 
Zeitg.,  1847,  p.  497,  ff.).— Mohl,  Die  veget.  Zelle,  p.  30,  etc. 
(c/*.,  auch  Wagner's  Handworterbuch,  Bd.  IV,  p.  189,  etc.). 
— Dippel,  Beitr.  z.  Losung  der  Frage,  etc.  (Bot.  Zeitg.,  1851, 
p.  409,  ff.)—  Schacht,  D.  Pflanzenzelle  a.  v.  O.,  z.  B.  p.  143, 
ff. — Pringsheim,  Algologische  Mittheilungen  (Flora,  1852,  p. 
470,  ff. ) — Hof'meister,  Ueb.  die  zu  Gallerte  aufqnell.  Zellen  der 
Aussenflache  v.  Samen  u.  Perikarpien  (Ber.  Kon.  Sachs.  Ges- 
ellsch.  der  Wiss.  Bd.  X,  1858,  p.  21,  ff.)  —  Frerny,  Recb. 
chim.  sur  la  compos,  des  Cellules  veget.  (Comptes  rendus,  t. 
XLVIII,  1859,  p.  202,/".)— Fremy,  Caracteres  distinctits  des 
fibres  lign.,  des  f.  corticales  et  du  tissu  cellulaire  qui  consiste 
la  moelTe  des  arbres  (1.  c.,  t.  XLVIII,  1859,  p.  275,  ff)  - 
Payen,  Differents  etats  de  la  cellulose  dans  les  pi.  (I.  c. ,  t. 
XLVIII,  1859,  p.  772, /".)— Mulder,  Physiol.  cheni.,  p.  475. 
— Kabsch,  Unters.  lib.  d.  chem.  BeschaiFenh.  d.  Zelhvande 
(Pringsheim's  Jahrb.,  Bd.  Ill,  1863,  p.  357,/".)—  Nageli,  Ueb. 
d.  Verhalten  d.  Zellhuut  zum  Jod.  (Sitzungsber.  d.  bayer. 


320  THE  MICROSCOPE  IN  BOTANY. 

Acad.  d.  Wiss.,  1863,  Bd.  I,  p.  383, /*.)— Nageli,  Uber  die 
Reactionen  von  Jod  auf  Starkekorner  u.  Zellmembr.  (L  c.,  p. 
483,/1.)— Hofmeister,  Handb.  d.  physiol.  Bot.  Bd.  I,  p.  252, 
/*.,  etc.— Sach's  Handb.  d.  Experi mental phys.  d.  Pfl.,  p.  433, 
ff. — Hofmeister,  D.  Lehre  v.  d.  Pflanzenzelle,  Lpz.,  1867,  a.  v. 
O. — Dippel,  Mikroskop,  Bd.  II,  p.  6,  ff. — Sach's  Lehrb.  d. 
Bot.  p.  19,^. — Nageli  und  Schvvendener,  Mikrosk.,  p.  474,  p. 
517, /.,  p.  549,  etc.— Strasburger,  Zellbild.  u.  Zellthiel.  III. 
Aufl.  1880,  a.  v.  O. — Ponlsen,  Botanisk  Mikrokemi,  p.  49,/*. 
(Trans,  p.  75). —  Richter,  Beitr.  z.  genaueren  Kenntniss  der 
chem.  Beschaffh.  der  Zellmembranen  bei  den  Pilzen  (Sitzungs- 
ber.  K.  K.  Acad.  d.  Wiss.  Wien,  Bd.  LXXXIII,  1  Abth., 
1881,  pp.  494-510). 

When,  as  appears  from  the  investigations  of  Strasburger,5 
in  the  dividing  of  a  cell,  the  primary  cell  plates  form  between 
the  connecting  fibres  of  the  separated  nuclei,  there  will  be  intro- 
duced into  these  by  intercalation  very  fine  granules,  which  by 
their  behavior  towards  iodine  solutions  (see  below)  will. demon- 
strate themselves  to  be  grains  of  starch  of  the  utmost  minute- 
ness.6 But  as  soon  as  the  granules  are  transformed  into  the 
substance  of  the  outer  cell  wall  they  will  then  give  no  reaction7 
whatever,  either  by  the  addition  of  chlor-iodide  of  zinc,  or 
iodine  and  sulphuric  acid.  These  primary  cell  plates  are  either 
transitory,  that  is  to  say,  are  absorbed  and  give  place  to  cellu- 
lose plates  or  they  coexist  with  these  and  are  employed  in  their 
formation.  . 

The  completed  but  very  young  cell  membranes  of  the  meres- 
tematic  tissue  are  not  often  colored  (Dippel),  or  if  at  alt  yellow 
(Solla)8  by  the  use  of  iodine  and  sulphuric  acid  or  chloi -iodide 
of  zinc.  If,  however,  they  are  previously  treated  with  muriatic 
acid,  or  potash  lye  or  have  lain  for  a  short  time  in  water  in  which 
the  process  of  fermentation  is  taking  place  they  would  be  col- 
ored blue  by  a  brief  subjection  to  the  influence  of  the  iodine 
reagent.9 

6  Strasburger,  Zellbild.  und  Zellth.  Ill,  Aufl.,  1880,  p.  1,  ff. 
8  Strasburger,  I.  c..  p.  16,  Table  I,  Fig.  6-9. 

7  Strasburger,  L  c.,  p.  13. 

»  Dippel,  Mikrosk.,  Bd.  II,  p.  7,/.  — Solla  in  Oesterr.  Bot.  Zeitschr.  Jahrg.,  1879,  p.  351. 
»  Richter  in  Sitzungsber.  der  K.  K.  Acad.  d.  Wiss.  Wien,  Bd.  LXXXIII,  1  Abth.,  1881,  p. 
498. 


CELLULOSE  AND  IODINE  KEAGENTS.  321 

The  older  cell  layers,  which  consist  of  pure  cellulose,  are  not 
colored  at  all,  or  only  with  a  yellowish  or  brown -yellowish  or 
reddish  tint  by  the  addition  of  freshly  prepared  iodine  water.10 
But  if  the  iodine  water  contain  traces  of  hydriodic  acid  a  blue 
or  violet  color  will  be  produced  (Nageli).  But  if  the  prepar- 
ation which  has  been  impregnated  with  iodine  water  he  treated 
to  a  drop  of  sulphuric  acid  or  caustic  potash,  there  immediately 
appears  an  intense  blue  color,  while  neither  muriatic  nor  nitric 
acid  will  produce  this  staining  (Meyer,  Schleiden).  Chlor- 
iodide  of  zinc  solution  colors  pure  cellulose  blue  under  all 
circumstances  and  is  the  most  important  reagent  for  it.  Zinc 
chloride  causes  the  cell  walls  to  swell  very  quickly  and  so 
disturbs  their  natural  relations  to  each  other.  This  can,  how- 
ever, be  prevented  for  a  considerable  time  by  suitably  diluting 
the  fluid  with  water  or  potassium  iodide  of  iodine  solution. 
Chlor-iodide  of  zinc  is  almost  always  to  be  preferred  to  iodine 
water  and  sulphuric  acid,  since  the  sulphuric  acid  very  quickly 
destroys  the  whole  tissue. 

The  intensity  but  not  the  shade  of  the  blue  or  violet  color 
which  is  produced  by  the  iodine  solution  is  conditioned  upon 
the  quantity  of  the  intercalated  iodine  in  the  membrane. 

^Nageli11  who  subjected  the  behavior  of  pure  cellulose  mem- 
brane towards  iodine  to  a  very  searching  investigation  reached 
the  following  principal  results. 

The  quantity  of  the  intercalated  iodine  determines  in  general 
not  the  character  but  only  the  intensity  of  the  color.  Each  tint 
(yellow,  orange,  red,  violet,  blue)  may  be  made  bright  by  less 
iodine  and  intense  by  the  use  of  a  greater  quantity.  One  may 
observe  in  single  cases  a  transition  from  bright  yellow  to  dark 
blue  when  during  the  reaction  of  the  iodine  hydriodic  acid  is 
formed.  In  other  cases  the  absorption  of  more  iodine  changes 
the  color  from  blue  to  brown  when  the  membrane  consists  of 

10  The  walls  of  the  spore  utricle  of  the  lichens  are  an  exception  to  this,  however,  as 
they  are  colored  blue  by  the  use  of  iodine  water  alone.    (Xageli  in  Sitzungsber.   Bayer. 
Acad.,  18C3,  Bd.  I,  p.  485,  ff.},  the  blue  color  becomes,  however,  more  intense  by  addition 
of  sulphuric  acid  (Kichter,  I.  c.,  p.  496).    For  the  rest  see  also  Mohl  in  Flora,  1840.  II  Bd. 
p.  614,  and  G.  Dickie  in  Annals  of  Nat.  Hist.,  1839,  p.  165.— Concerning  the  reactions  of  the 
cellulose  walls  of  spores  of  algae,  see  Pringsheim  in  Flora,  1852,  p.  470, /. 

11  Xiigeli,  concerning  the  reactions  of  iodine  on  starch  grains  and  cell  membranes    (Sit- 
zungsberichte  der  Bayerischen  Acad.  1863,  Bd.  I,  pp.  524,  530,  532,  535,  539,  541,  543). 

21 


322  THE  MICROSCOPE  IN  BOTANY. 

a  mixture  of  two  different  materials  which  are  acted  upon 
differently  by  the  iodine. 

Cell  membranes  which  are  permeated  by  water,  and  have 
received  some  one  color  by  iodine,  retain  this  color  when  the 
water  is  drawn  out  of  it  at  the  common  temperature,  and  when 
otherwise  no  chemical  or  physical  change  has  occurred.  But,  on 
the  contrary,  if  some  substance  is  dissolved  by  the  interpenetrat- 
ing water  which  is  again  concentrated  by  the  evaporation,  it  may 
so  effect  the  arrangement  of  the  molecules  of  iodine  as  to  cause 
a  greater  or  less  change  of  color. 

Membranes  colored  by  iodine,  which  may  become  'uncolored 
either  in  the  moist  or  dry  condition,  frequently  change  their 
color  more  or  less.  These  transformations  always  proceed  in 
the  direction  from  blue  through  red  to  yellow. 

When  a  cell  membrane  will  not  immediately  stain  by  iodine 
and  water  it  may  be  colored  by  the  action  at  the  same  time, 
of  hydriodic  acid  (which  is  formed  by  the  prolonged  action  of 
iodine  on  different  organic  combinations  as  well  as  by  drying 
them  with  iodine),  or  of  potassium  iodide,  or  ammonia  iodide, 
zinc  iodide,  phosphoric  acid  or  sulphuric  acid,  in  other  cases 
also  by  sulphuric  acid  after  a  more  or  less  energetic  treatment  by 
caustic  potash  or  nitric  acid. 

The  treatment  with  hydriodic  acid,  potassium  iodide,  am- 
monium iodide,  with  sulphuric  and  phosphoric  acid,  caustic 
potash  and  nitric  acid  removes  without  doubt  a  less  or  greater 
quantity  of  foreign  substances  contained  in  the  membrane  which 
are  soluble  in  that  particular  combination.  This  purifying 
of  the  cell  membrane  may  in  many  cases  facilitate  the  bluing, 
but  it  is  in  no  case  the  only  determining  condition  of  it. 

Treatment  by  the  above  named  reagents  causes  a  greater  or 
less  swelling  of  the  membrane  but  this  loosening  up  of  the  tis- 
sue is  in  no  case  the  cause  of  the  bluing. 

For  the  bluing  of  the  cell  membrane  with  iodine  and  water 
(except  in  the  case  of  the  lichen  tissue)  there  is  required  the 
presence  at  the  same  time  of  an  assisting  combination,  hydriodic 
acid,  potassium  iodide,  ammonium  iodide,  zinc  iodide  (or  an- 
other metallic  iodide),  sulphuric  acid,  phosphoric  acid,  zinc 
chloride  (?).  But,  perhaps,  the  sulphuric  and  phosphoric  acid 


CELLULOSE  AND  MINERAL  ACIDS.  323 

do  not  act  directly  but  indirectly  by  favoring  the  formation  of 
hydriodic  acid,  through  the  decomposition  of  alcohol,  or  of  or- 
ganic combinations  of  the  cell,  so  that  thence  the  blue  color  is 
almost  exclusively  conditioned  by  the  presence  of  a  definite 
quantity  of  an  iodine  combination. 


B.     Behavior  of  Cellulose  towards  Mineral  Acids. 

Of  these,  concentrated  sulphuric  and  chromic  acid  are  partic- 
ularly adapted  to  give  the  wished-for  demonstration.  In  opposi- 
tion to  hydrochloric  acid  which  leaves  the  cellulose  almost 
perfectly  unchanged,  and  to  nitric  acid  which  when  cold  causes 
only  a  swelling  of  the  cell  membrane  and  dissolves  it  only  by 
boiling,  the  other  acids  named  dissolve  the  cellulose  at  ordinary 
temperature  and  in  a  very  short  time.  On  the  contrary  the 
modifications  of  cellulose,  middle  lamella,  suberin,  together 
with  cutin  are  not  soluble  in  them  (see  below).  However,  it 
should  be  stated  that  (probably  all)  wood  membranes  share  with 
cellulose  in  the  narrow  sense,  its  solubility  in  sulphuric  and 
chromic  acid.  If  a  section  through  the  stem  of  a  plant  be  put 
into  concentrated  sulphuric  acid  all  that  part  which  consists  of 
lignin  and  pure  cellulose  will  be  dissolved  out,  while  suberized 
layers  and  cuticularized  combinations,  as  well  as  the  middle 
lamella  of  the  woody  tissue,  will  remain  behind  intact.  Cellu- 
lose is  changed  by  sulphuric  acid  into  an  isomeric  body  to  which 
Schleiden  gave  the  name  amyloid,  because  while  it  in  itself 
remains  unchanged  it  effects  changes  in  other  bodies  by  con- 
tact, or  "catalysis"  as  the  chemists  say.  This  amyloid  stands  very 
near  to  starch  as  we  infer  from  the  fact  that  by  the  application1 
of  iodine  water  it  takes  on  a  very  intense  blue  color.  On  this 
circumstance  rests  the  above  many-times-mentioned  reaction  of 
iodine  and  sulphuric  acid  on  cellulose,  and  is  conducted  in  the 
following  waj'.  Put  the  section  to  be  examined  in  freshly  pre- 
pared iodine  water  for  a  short  time.  Take  it  out  and  remove  as 
much  as  possible  of  the  adhering  fluid,  lay  it  on  a  slide,  put  on 
a,  cover  glass,  put  the  preparation  under  the  microscope,  and 
add  at  the  edge  of  the  cover-glass  a  large  drop  of  concentrated 


324  THE  MICROSCOPE  IN  BOTAN1T. 

sulphuric  acid.  Now  look  quickly  in  the  microscope  and  see 
how  all  the  true  cellulose  membranes  take  on  an  intense  blue 
color,  while  all  the  modifications  of  cellulose,  lignin,  middle 
lamella,  and  suberin  are  stained  yellow  or  brown.  After  a  few 
moments,  however,  the  blue  reaction  becomes  indistinct  because 
of  the  destruction  of  the  tissue  which  goes  rapidly  forward. 


C.     Behavior  of  Cellulose  toiuards   Alkalies. 

In  comparison  with  the  destructive  effect  of  mineral  acids 
upon  cellulose,  the  alkalies  take  but  little  if  any  hold  upon  it. 
Ammonia  itself  even  in  a  concentrated  state,  and  when  the  sec- 
tion is  boiled  in  it  for  a  short  time,  docs  not  alter  the  substance 
of  the  cell  walls,  while  concentrated  or  nearly  concentrated 
potash  lye  will  only  cause  them  to  swell.  By  washing  out  the 
section  treated  with  potash,  and  putting  it  in  absolute  alcohol 
the  cell  walls  will  resume  their  original  form,  on  which  account 
Hanstein's  method  of  bleaching  is  recommended  (see  p.  199)  ; 
compare  also  what  is  said  concerning  Russow's  potassium  alco- 
hol. 

D.     Behavior  of  Cellulose  towards  Cuprammonia. 

Schweizer12  found  in  1857  that  cotton  laid  in  cuprammonia 
very  quickly  dissolved  and  assumed  a  jelly-like  consistency, 
thence  changed  into  a  mucilaginous  fluid  which  on  being  diluted 
with  water  was  filtered.  On  applying  muriatic  acid  a  precipitate 
was  thrown  down  which  was  colored  brown  by  potassium  iodide 
and  chlorine  water,  showing  that  the  cellulose  was  not  changed 
to  starch  by  the  process.  Cellulose  is  distinguished  from  all 
its  modifications  by  the  characteristic  of  its  being  soluble  in  cu- 
prammonia. Put  a  drop  of  the  freshest  possible  preparation 
of  the  reagent  upon  a  damp  section  lying  under  the  cover- 
glass  and  then  observe  how  the  cellulose  walls  gradually  swell 
up.  Afterwards  the  outlines  become  indistinct  and  the 


12  Schweizer,  Jour,  prakt.  Chem.,  Bd.  LXXII,  p.  Ill  —  see  also  above,  p.  244,  ff\,  then  the 
treatise  of  Fremy  and  Payen  cited  on  page  268. 


CELLULOSE  AND  CARMINE.  325 

cess  ends  with  a  perfect  solution  of  the  whole  cellulose  struct- 
ure. It  may  be  remarked  in  this  connection  that  the  modi- 
fications of  cellulose  may  be  dissolved  in  cuprammonia  when 
the  intercalated  incrusting  substance  has  previously  been  re- 
moved. This  is  most  readily  accomplished  by  Schnitzels  macer- 
ation process,  with  potassium  chlorate  and  nitric  acid  (see  p. 
163).  Boil  the  section  with  this  mixture  in  a  test  tube,  wash 
out  the  imdestroyed  portion  with  water  and  put  it  on  the  slide 
with  a  drop  of  cuprammonia  solution. 

E.     Behavior  of  Cellulose  towards  Alum  Carmine. 

Literature.  E.  Tangl,  Concerning  the  open  communication 
between  the  cells  of  the  endosperm  of  some  seeds  (Pringsheim's 
Jahrb.,  Bd.  XII,  1879-81,  p.  170,  f.) 

According  to  Tangl,  Grenadier's  alum  carmine  (p.  308)  offers 
a  superior  means  of  distinguishing  pure  cellulose  membrane  from 
that  which  has  been  cuticularized  or  changed  to'  cork.  For 
while  the  former  eagerly  takes  up  the  coloring  matter  and  after 
five  to  ten  minutes  becomes  an  intense  red,  the  latter  remains 
uncolored.  The  color  keeps  very  well  in  glycerine,  and  gives 
us  a  very  instructive  specimen  and  particularly  so  in  respect  to 
the  vascular  bundles.  In  order  to  get  a  beautiful  stain  of  the 
nucleus  and  plasma  one  should  previously  harden  the  prepara- 
tion in  absolute  alcohol  (see  above  p.  308). 13 

F.     Behavior  of  Cellulose  towards  Potassium- Copper 
Sulphate. 

Literature.  Sachs,  Concerning  some  new  methods  of  micro- 
chemical  reactions  (Sitzungsberichte  der  K.  acad.  d.  Wiss.,Bd. 
XXXVI,  185(J,  p.  1-22).  Sachs,  Micro-chemical  investiga- 
tions (Flora,  1862,  p.  288,  /".).  Sachs,  Concerning  the  sub- 
stances which  furnish  the  material  for  the  growth  of  the  cell 
wall  (Pringsheim's  Jahrb.,  Bd.  Ill,  1863,  p.  187,  ff.). 

13  Tangl  states,  I.  c.,  p.  173,  also  that  the  cellulose  membrane  of  cambium  and  older 
parenchyma  cells  absorbs  the  blue  coloring  matter  from  an  aqueous  decoction  of  logwood 
in  insoluble  modification  if  this  contains  an  addition  of  sulphate  of  iron  and  is  applied  cold. 
However,  the  same  thing  may  happen  in  modified  cellulose  membrane  so  that  it  will  not 
serve  as  a  test  of  cellulose. 


326  THE  MICROSCOPE  IN  BOTANY. 

Sachs  has  given  a  process  of  bluing  membranes  which  con- 
tain a  certain  kind  of  cellulose  by  treatment  with  copper  sul- 
phate and  potassium  solution.  The  method  is  as  follows.  A 
very  thin  section,  if  possible  thinner  than  the  thickness  of  a 
layer  of  cells,  should  be  laid  for  a  long  time  in  a  concentrated 
solution  of  copper  sulphate.  It  should  remain  in  this  from  five 
to  ten  minutes,  or  several  hours,  or  a  day  even,  according  to  its 
nature.  Then  remove  the  section  and  lay  it  for  a  few  minutes  in 
water  in  order  to  wash  off  the  salt  solution.  For  this  purpose  it 
is  necessary  to  put  the  section  in  a  considerable  quantity  of  water 
and  not  merely  in  a  drop  on  the  slide.  The  better  way  is  to 
take  the  section  in  the  forceps  and  move  it  back  and  forth  sev- 
eral times  in  pure  water.  Then  in  a  porcelain  saucer  which 
holds  8  or  10  cc.  make  a  strong  potassium  solution  of  1  part 
by  weight  of  water,  and  1  part  caustic  potash,  and  put  the  sec- 
tion in  it  for  a  short  time,  till  in  certain  of  the  cell  walls  there 
appears  a  blue  tint  and  in  others  a  yellow  color,  while  others 
still  remain  colorless.  Lying  in  a  drop  of  the  fluid  the  section 
can  then  be  examined.  Then  put  the  section  in  a  small  porce- 
lain cup  and  boil  it  for  a  few  minutes  in  the  potash  solution. 
This  intensifies  the  color  and  makes  it  appear  now  for  the  first 
time  general.  If  the  color  is  too  transparent  one  must  take 
a  section  sufficiently  thick  to  make  it  distinct  and  charac- 
teristic. 

By  this  method  many  but  not  all  cell  walls  consisting  of  cell- 
ulose will  be  colored  an  intense  blue,  while  others  will  remain 
colorless.  Those  cell  walls  which  are  colored  yellow  with  iodine 
are  not  cellulose  in  the  strict  'sense  and  are  colored  yellow  or 
orange  yellow  by  this  method.  So,  according  to  Sachs,  the 
peripheral  layer  of  parenchyma  of  the  germ-root  of  the  horse 
bean  (  Vicia  faba)  takes  on  a  beautiful  blue  stain  ;  also  parts  of 
the  parenchyma,  the  very  }roung  vascular  bundles,  wood  cells, 
and  bast  cells  of  the  germ-root  of  Phaseolus  multiflorus,  the 
subepidermal  layer  from  the  blooming  branch  of  the  gourd, 
and  generally  all  collenchyma  cells,  young  bast  cells  and  wood 
cambium  become  blue,  while  commonly  the  thick  walled  par- 
enchyma cells  remain  uncolored.  The  walls  of  older  bast  cells 
color  yellow  or  yellow  orange,  and  all  lignified  elements  of  the 


MUCILAGINOUS  CELLULOSE.  327 

wood  bodies.14  Since  a  characteristic  reaction  will  not  take 
place  in  all  cellulose  walls  by  this  method,  its  application  there- 
fore is  not  a  sufficient  test  without  verification  by  iodine  reac- 
tions. 

2.    MUCILAGINOUS  CELLULOSE. 

Literature.  Meyen,  Die  Secretionsorgane  der  Pflanzen,  Ber- 
lin, 1837,  p.  36,  etc.  —  Schleiden  in  Wiegmann's  Archiv,  1838, 
p.  145. — Deciasne,  Sur  la  structure  des  poils  qui  couvrent  le 
pericarpe  de  certaines  composees  (Ann.  des  Soc.  Nat.  lie,  Ser. 
t.  XII,  1839,  pp.  251-254).— Meycn,  Pflanzpathologie,  Berlin, 
1841,  p.  235.  — Schleiden,  Beitr.  z.  Botanik  (1844),  p.  137. 
— Mohl,  Einige  Bemerk.  fiber  den  Ban  der  vegetal).  Zelle 
(Botan.  Zeitung,  1844,  p.  323,  /.).— Mohl,  Yermischte  Schr. 
a.  a.  O. — Kippist,  On  the  existence  of  spiral  cells  in  the  seeds 
of  AcanthncejB  (Trans,  of  the  Linn.  Soc.  of  London,  1845, 
Vol.  XIX,  pp.  65-76). —  Kutzing,  Grundziige  der  philosoph. 
Botanik,  Leipzig,  1852,  Bd.  I,  p.  195,  ff.  —  Cramer,  Botan. 
Beitrage,  Zurich,  1855,  p.  1,  ff. —  Unger,  Anat.  und  Physiol. 
der  Pflanzen,  Pest,  1855,  p.  78,  119,  Taf.  IV. — Karsten, 
Ueber  die  Entstehung  des  Harzes,  TVachses,  Guminis,  und 
Schleinies  durch  die  assimilirende  Thatigkeit  der  Zellmem- 
branen  (Bot.  Zeitg.,  1857,  p.  313,  ff.).— Mohl,  Ueber  die 
Entstehungsweise  des  Traganthgummi  (Bot.  Zeitg.,  1857,  p. 
33,  ff.) — Hofmeister,  Uber  die  zu  Gallerte  aufquell.  Zellender 
Aussenflache  von  Samen  und  Perikarpien  (Berichte  der  Sachs. 
Gesellschaft  zu  Leipzig,  Bd.  X,  1858,  pp.  18-36).— Trecul,  in 
Comptes  rendus,  1860;  Journal  de  1'Institut,  1862,  p.  241.- 
Wigand,  Ueber  des  Desorganisation  der  Pflanzenzelle,  insbes. 
fiber  die  physiol.  Bedeutung  von  Giunmi  und  Harz  (Pringsheim's 
Jahrb.,  Bd.  Ill,  1863,  pp.  155-182).— Frank,  Uber  die  anat. 
Bedeut.  und  die  Entstehung  der  veget.  Schleime  (Pringsheim's 
Jahrb.,  Bd.  Y,  1866,  pp.  161-200).— Hofineister,  Handb.  d. 
physiol.  Botan.,  Bd.  I  (1867),  p.  258,  ff.—  Hanstein,  Ueber 
die  organe  der  Harz-  und  Schleimabsonderung  in  den  Laub- 
knospen  (Botan.  Zeitg.,  1868,  p.  697,  ff.) — Behrens,  Unters. 
fiber  den  anat.  Ban  des  Griflfels  und  der  Narbe,  Gott.,  1875,  p. 

"  Sachs,  in  Sitznngsber.   Wein,  1.  c.,  p.  18, 19,    Flora,  1862.  p.  295. 


328  THE   MICROSCOPE  IN  BOTANY. 

28,  jf. —  Prillieux,  Etude  sur  la  formation  do  la  gomme,  etc. 
(Ann.  des  sc.  nat.  Vie  ser,  t.  I,  1875,  pp.  176-200). —  Reinke, 
Beitr.  zur  Anat.  der  an  Laubblattern,  besond.  an  den  Zahnen 
ders.  vorkoram.  Sccretionsorgane  (Pringsheim's  Jahrb.,  Bd.  X, 
1875,  pp.  119-178). —  Capns,  Anatomic  du  tissn  conductenr 
(Ann.  des  sc.  nat.  Vie  ser.,  t.  VII,  1878.)—  Behrens,  Die 
Nectarien  der  Bliiten  (Flora,  1879,  p.  118,  f.,  144,  /".,  233, 
jf.,  440,  ff.). — Dalmer,  Ueber  die  Leitnng  der  Pollensehlauche 
bei  clen  Angiosperrnen  (Jenaische  Zeitschr.,  /.,  Naturwissen- 
schaft,  Bd.  XLV,  1880,  a.  a.  O.). 

Meyen  and  linger  have  already  observed  the  fact  that  cer- 
tain cells  and  cell  groups  whose  walls  originally  consisted  of  true 
cellulose  were  gradually  transformed  by  a  process  of  degen- 
eration into  isomeric  carbo-hydrate  combinations  which  in  their 
chemical  but  principally  in  their  physical  behavior  are  more  or 
less  different  from  cellulose.  Although  the  finished  product  of 
the  mnciparous  process  cannot  be  any  longer  regarded  as  cel- 
lulose (it  will  be  treated  more  in  detail  under  the  section 
"vegetable  mucilage"),  yet  in  the  first  stages  of  the  transfor- 
mation of  cellulose  to  mucilage  it  is  so  much  like  cellulose  that 
it  should  be  described  in  this  place.  The  cellulose  walls  of  the 
inuciparous  cells  and  cell  groups  have  quite  generally  the  ca- 
pacity of  swelling  to  several  times  their  original  volume  by  an 
unusual  power  of  imbibing  water;  thus  often,  the  whole  cell, 
and  always  the  inuciparous  tissue,  loses  its  original  form  and 
is  changed  into  a  quite  thin  jelly.  If  the  muciparous  cell  walls 
are  thickened  and  stratified  as  are  those  of  the  Astragalus™  spe- 
cies whose  medullary  rays  furnish  the  gum  tragacanth,  the  strat- 
ification will  gradually  become  indistinct  till  at  last  the  resulting 
mucilaginous  mass  will  appear  to  be  very  nearly  structureless.16 
The  whole  of  the  cell  wall  or  only  its  outermost  layer  may  be 
drawn  into  sympathy  with  this  process  and  it  ends  with  its 
transformation  into  a  jelly  more  or  less  soluble  in  water.17  In 
other  cases  a  middle  portion  of  the  wall,  which  is  often  strongly 
developed,  is  dissolved  into  a  fluid  amyloid  (Collagen)  as  for 
example  in  epidermal  cells  where,  by  the  absorption  of  water, 

«  Mohl,  in  Botan.  Zeitung,  1857,  p.  33,^". 

16  Compare  Prillieux  in  Ann.  sc.Nat.,  Vie  Ser.,  1. 1,  pi.  V.  fig.  1. 

"Mohl,Z.  c.,  p.  42,  /. 


MUCILAGINOUS  CELLULOSE.  329 

they  swell  out  and  become  a  mucilaginous  jelly  raising  up  the 
cuticle  into  blisters  which  finally  burst.18  In  such  cases  there 
remains  commonly  besides  the  cuticle  a  thin  layer  of  cellulose 
not  participating  in  the  process  of  disorganization,  which  sepa- 
rates the  mucilaginous  complex  from  the  interior  of  the  cell ;  but 
it  is  always  a  part  of  the  strongly  thickened  cell  wall  which  swells 
up  to  become  mucilage,  the  process  itself  being  much  diversi- 
fied in  details. 

The  swollen  substance  sometimes  agrees  with  cellulose  both 
in  its  anatomical  behavior  and  its  chemical  reactions.  The  jelly 
under  discussion  is  colored,  for  example,  blue  (Hofmeister)  with 
iodine  water  and  iodine  alcohol  in  the  seeds  of  Salvia  horminum 
and  Teesdalia  nudicaulis  or  pale  blue  (seeds  of  Linum  usitatis- 
simum).  Iodine  in  connection  witj)  sulphuric  acid  of  a  certain 
definite  concentration  for  each  individual  case  colors  it  blue.  The 
swollen  layer  of  the  seeds  of  Salvia  horminum  blued  by  iodine 
becomes  reddish19  by  the  addition  of  dilute  sulphuric  acid.  In 
other  cases  the  muciparous  cellulose  tissue  behaves  quite  dif- 
ferently towards  iodine  reagents,  often  not  taking  any  color 
whatever  from  any  kind  of  iodine  combination  (Collagen,  Plan- 
stein).  Where,  as  in  the  formation  of  gum  tragacanth,  the 
muciparous  cell  walls  originally  showed  color  with  chlor-iodide 
of  zinc  solution,  this  reaction  becomes  weaker  in  the  mass  as  the 
muciparous  process  goes  on  till  finally  it  altogether  ceases 
(Mohlj.  Other  mucilaginous  substances  take  a  yellow  color 
with  iodine  and  sulphuric  acid  and  with  chlor-iodide  of  zinc;20 
only  rarely  do  the  older  stages  of  muculent  complex  give  the 
reaction  of  cellulose,  viz.,  bluing  by  the  two  well-known  re- 
agents.21 So  the  reagents  show  with  their  different  effects,  that 
in  the  process  under  discussion  we  have  detected  all  transition 
from  true  cellulose  to  true  mucilaginous  matter. 

As  the  most  successful  method  of  investigating  muciparous  cells 
the  following  may  be  mentioned.  According  to  the  statements 
of  Fenzl22  sections  through  muciparous  seeds  are  best  made  by 

"  Hanstein  in  Bot.  Zeit.,  1868,  p.  697  ff.—  Behrens  in  Flora,  p.  118,  ff.t  p.  232,  ff. 

19  Hofmeister  iu  Ber.  Sachs,  Ges.  Leipzig,  Bd.  X,  p.  30. 

2°  Frank  in  Pringsheim's  Jahrb.,  Bd.  V,  pp.  163,  165, 167,  etc. 

21  Kutzing.  Grundz.  d.  philos.  Bot.,  Bd.  I,  p.  195.— Frank,  1.  c.,  pp.  168, 181. 

22  Feuzl  in  Dcnkschr.  d.  Wciner  Acad.  VIII,  Erkl.  zu.  Taf.  I. 


330  THE  MICROSCOPE  IN    BOTANY. 

embedding  them  in  stearine  ;  having  cut  the  section  wash  in  al- 
cohol to  remove  any  shavings  of  stearine.  The  embedding  me- 
dium described  on  pp.  186-7,  given  by  Koch,  may  be  employed. 
The  section  should  be  first  studied  dry  or  better  still  in  alcohol, 
sometimes  also  in  essential  oils,  because  the  use  of  water  causes 
a  rapid  swelling.  Since  very  delicate  mucilaginous  substances 
refract  the  light  quite  like  alcohol  and  therefore  can  with  diffi- 
culty be  recognized  in  it,  a  small  quantity  of  coloring  matter, 
which  will  not  be  absorbed  by  the  mucilage,  should  be  added 
to  the  alcohol23  in  such  cases.  For  this  purpose  nothing  is  bet- 
ter than  the  aniline  dyes.  If,  after  having  studied  the  prepara- 
tion in  alcohol,  water  is  added,  the  muciparous  cells  rapidly 
expand  in  a  radial  direction,  at  the  same  time  the  mucilaginous 
complex  begins  to  swell  very  quickly,  the  swollen  substances 
immediately  separate  in  the  water  and  directly  disappear  from 
observation.  The  addition  of  potassium  solution  will  produce 
a  swelling  in  the  dry  preparation. 


3.     WOOD   SUBSTANCE.     (Lignin.) 

Wood  or  lignin  (C19  H24  O10)24  forms  the  walls  of  all  those  cells 
which  are  changed  into  wood.25  Lignified  cells  are  commonly 
found  in  wood  bodies;  other lignified  cells  are,  however,  found 
isolated  in  the  parenchyma  tissue,  as  for  example,  the  stone- 
cells  which  occur  in  the  pith  of  numerous  woody  plants ;  cells 
of  like  name  in  the  pulp  of  the  fruit  of  the  Pomacece  and 
similar  cell  tissue  in  the  bark  layer,  etc. 

In  all  lignified  cell  walls,  as  mentioned  on  p.  316,  there  is  inter- 
calated an  incrusting  substance,  which  contains  relatively  more 
hydrogen  and  carbon  than  does  cellulose  in  the  strict  sense. 
According  to  the  later  investigations  of  Singer  and  the  earlier 

o  o  o 

ones  of  v.  Hohnel  there  are  four  substances  which  constantly 

23  Hofmeister,  1.  c.,  p.  21. 

24  Particulars  in  Burgerstein  in  proceedings,  d.  K.  Acad.  Wein,  Bd.  LXX,  1  Abth.,  1871, 
p.  338,  Anm. 

SB  FOI.  generai  ijsts  of  literature  see,  Sachsse,  Chemie  mid  Physiol.  der  Farbstoffe,  Car- 
bo-hydrates u.  Proteinsubst.  Lpz.,  1887,  p.  144,  ff.—  Niggl,  Ueber  die  Verholzung  d.  Zell- 
membranen  (Jahresber.  Pollichia,  1881).— Ebermeyer,  Physiol.  Chem.  der  Pflanzen,  Berlin, 
1882,  Bd.  I,  p.  174,  JT.— Singer,  Beitrage  ztir  naheren  Kenntniss  d.  Holzsubstanz,  u.  d.  ver- 
holzt.  Gewebe  (Silzuiigsber.  d.  K.  Acad.  Wien,  Bd.  LXXXV,  1882, 1  Abth.,  p.  345,  JO 


LIGNIN  AND  IODINE  EEAGENTS.  331 

accompany  lignified  tissues,  namely,  Vanillin,  Coniferin  and  a 
kind  of  gum  which  stands  near  to  Arabin  and  perhaps  repre- 
sents a  modification  of  the  wood  gum  of  Thompson,  and  fin- 
ally a  body  which  takes  a  yellow  color  with  muriatic  acid  but 
whose  chemical  nature  is  still  quite  unknown.  All  these  ele- 
ments may  be  extracted  by  boiling  the  wood  in  water  for  a 
longer  or  shorter  time  ;  they  are  that  which  gives  the  character- 
istic reactions  of  wood-substances.  In  what  relation  they  stand 
to  lignin  cannot  yet  be  made  out,  but  they  indicate  that  that 
which  we  call  lignin  represents  a  mixture  of  several  chemical 
individuals. 

As  the  most  important  reagents  for  lignified  cell  walls,  iodine 
solutions,  aniline  sulphate,  phloroglucin,  indol,  and  phenol- 
hydrochloric  acid  should  be  named. 

As  a  distinction  from  pure  cellulose,  lignin  does  not  dissolve 
in  cuprammonm.  It  is,  on  the  contrary,  soluble  in  potash  lye 
(more  easily  than  cellulose)  concentrated26  nitric  acid,  sulph- 
uric and  chromic  acid.  Concentrated  sulphuric  acid  blackens 
it  in  dissolving.  Schulze's  maceration  mixture  dissolves  lig- 
nin very  easily.27 

A.     Behavior  of  Lignin  towards  Iodine  Reagents. 

Literature.  See  the  treatises  cited  on  p.  319.  Also  Mohl, 
Einige  Bemerk.  liber  die  Ban,  der  vegetal).  Zelle  (Botan.  Zeitg., 
1844^  p.  307, /*.).— Schacht,  Lehrb.  d.  Anat.,  etc.,  Bd.  I,  p. 
16,  etc. — Fremy,  Kecherches  sur  la  comp.  chim.  du  bois 
(Comptes  rendus  de  Paris,  t.  XLVIII,  1859,  p.  862,  /".)— 
Payen,  Compos,  de  Fenveloppe  des  pi.  et  des  tissus  ligneux 
(Comptes  rendus  de  Paris,  t.  XLVIII,  1859,  p.  893, /".)— Sa- 
il io,  Einige  Bemerk.  liber  d.  Ban  des  Holzes  (Botan.  Zeitg. 
1860,  p.  193,  ff.) — Sanio,  Vergl.  Unters.  liber  die  Elementar- 
organe  d.  Holzkorpers  (Botan.  Zeitg.,  1863,  p.  85,^.) — Sanio, 

26  Weak  acids  frequently  make  the  layers  of  the  cell  wall  come  out  very  distinctly. 
Dilute  sulphuric  acid  colors  the  young  layers  of  lignifled  membranes  a  beautiful  i-ose  red 
(Hartig,  But.  Zeit.,  ISon.  p.  213). 

117  According  to  S:inio  (Dot.  Zeig..  1860,  p.  204),  the  wood  substance  is  disintegrated  into 
a  granular  mass  which  finally  comes  into  the  cell  cavity,  and  if  potash  be  now  added  this 
granular  mass  will  be  dissolved  in  it  with  yellow  color. 


332  THE  MICROSCOPE  IN  BOTANY. 

Yergl.  Unters.  iiber  die  Zusammensetz.  des  Holzkorpers  (Botan. 
Zeitg.,  1863,  p.  358,  ff.— Mulder,  Physiol.  Chcrn.^  Bd.  I,  p. 
209.— Dippel,  D.  Mikroskop,  Bd.  II,  p.  96,  ff.— Sanio,  Zur 
Anatomie  der  gem.  Kiefer  (Pringsheim's  Jahrb.,  Bd.  IX,  p. 
65,^".). — Sachs,  Lehrbuch  d.  Botanik,  p.  35. 

Lignified  cell-walls  are  colored  yellowish,  yellow  or  brownish 
by  the  application  of  any  of  the  iodine  reagents,  potassium 
iodide  of  iodine,  chlor-iodide  of  zinc,  and  iodine  and  sulphuric 
acid.  The  last-named  shade  is  produced  only  by  iodine  and 
sulphuric  acid,  or  very  rarely  by  chlor-iodide  of  zinc ;  in  this 
case,  however,  it  shows  only  a  brownish-yellow.  The  brighter 
and  darker  layers  which  are  commonly  perceptible  in  lignified 
walls  either  show  no  difference  on  the  application  of  iodine  and 
sulphuric  acid,  or  else  a  distinction  is  apparent  in  the  alternating 
stronger  and  weaker  yellow  color. 

Pure  yellow  is  seen  in  the  perfectly  lignified  portion  of  the 
wall,  while  such  parts  as  have  been  imperfectly  lignified  are 
shown  by  the  mixed  colors.  They  are  either  a  transition  be- 
tween yellow  and  blue  (blue-green  or  yellow-green)  or  reddish. 
The  yellow  color,  produced  by  iodine  reagents,  which  indicates 
lignification,  shows  itself  through  the  whole  extent  of  the  cell- 
wall,  or  the  wall  will  be  but  partially  colored  yellow.  In  this 
case  it  is  commonly  the  peripheral  layer  of  the  wall  which  has 
the  highest  degree  of  lignification,  while  the  inner  portion  next 
to  the  cell  space  is  often  less  lignified. 

The  layer  of  the  lignified  cells  which  lies  naked  about  the 
cell-cavity,  and  which  is  commonly  an  optically  distinguishable 
thickening  layer,  called  by  Sachs  "an  inner  shell,"  and  by  Dip- 
pel  a  tertiary  membrane,  behaves  very  differently.  This  is 
colored  yellow  by  the  use  of  iodine  and  sulphuric  acid  or  chlor- 
iodide  of  zinc  quite  infrequently  (Sanio)28,  but  also  reddish  yel- 
low, commonly  violet,  bluish  or  quite  blue,  consisting  in  these 
cases  of  slightly  lignified  or  quite  unlignified  cellulose.  Thus, 
for  example,  the  innermost  thickening  layer  of  the  wood  cells 
of  Pinus  sylvestris  consists  of  pure  cellulose. 

The  incrusting  substance  may  be  removed  from  all  lignified 
membranes  by  maceration,  so  that  the  cellulose  which  is  left 

28  Sanio  in  Botan.  Zeit.,1860,  p.  202. 


LIGNIN  AND  ANILINE  SULPHATE.  333 

will  give  the  characteristic  reaction  with  iodine  reagents  (see  p. 
317  and  p.  319, /*.)• 

Method.  Prepare  the  thinnest  possible  section  of  the  wood- 
tissues  to  be  examined,  and  impregnate  it  if  one  is  to  produce 
the  reaction  with  iodine  and  sulphuric  acid  (see  p.  323)  with 
potassium  iodide  of  iodine  or  alcohol  iodine,  by  laying  it  in  a 
dish  filled  with  these  solutions  for  a  longer  or  shorter  time.  The 
adhering  solution  should  be  washed  off  with  distilled  water,  and 
the  section  laid  upon  the  slide  and  a  cover-glass  put  over;  then 
a  drop  of  concentrated  sulphuric  acid  added  and  the  reaction  is 
quickly  observed.  In  most  cases  this  reaction  is  preferable  to 
that  by  chlor-iodide  of  zinc.  In  the  use  of  the  latter  reagent 
the  moist  section  must  sometimes  remain  in  the  solution  several 
hours  before  the  wished-for  reaction  will  take  place. 


B.     Behavior  of  Lignin  toicards  Aniline  Sulphate. 

Literature.  Runge,  in  Poggendorfs  Ann.,  Bd.  XXXI, 
1834,  p.  65. — Schapringer  in  Wochenschr.  d.  niederosterr. 
Gewerbevereins,  Bd.  XXVI,  p.  326. — Wiesner  in  Karsten's 
Botan.  Unters.,  1866,  Bd.  I,  p.  120.— Wiesner  in  Sitzungsber. 
derK.  Acad.  Wien,  Bd.  LXII,  1.  Abth.,  1870,  p.  202  (Sep- 
aratabdr.,  p.  32). — Wiesner,  Die  Rohstoffe  des  Pflanzen- 
reiches. — Burgerstein,  Unters.  iiber  d.  Yorkommen  u.  die 
Entsteh.  des  Holzstoffes  in  den  Geweben  d.Pfl.  (Sitzungsber.  d. 
K.  Acad.  Wien,  Bd.  LXX,  1.  Abth.,  1874,  pp.  338-355).— 
Hohnel,  Ueber  Kork.  mid  verkorkte  Gewebe  Uberhaupt  (id., 
Bd.  LXXVI,  1.  Abth.,  1877,  p.  527).— Hohnel,  Histochem. 
Unters.  iiber  d.  Xylophilin  u.  d.  Coniferin  (id.,  p.  663,  ff.,  u. 
a.  a.  O.). — Sachs,  Ein  Beitr.  z.  Kenntniss  des  aufsteigenclen 
Saftstromes  in  transpirirenden  Pflanzen  (Arb.  d.  Botan.  Insti- 
tutes zu  Wurzburg,  Bd.  II,  Hft.  1,  1878,  p.  150,  f.)—  Gau- 
nersdorfer,  Beitrage  z.  Kenntniss  der  Eigenschaften  mid 
Entstehung  des  Kernholzes  (Sitzungsber.  d.  K.  Acad.  Wicn, 
Bd.  LXXXY,  1882,  1.  Abth.,  pp.  9-41;.— Singer,  Beitrage  z. 
naheren  Kenntniss  der  Holzsubstanz  und  der  verholzten  Ge- 
webe (id.,  pp.  345-360). 


334  THE  MICROSCOPE  IN  BOTANY. 

It  was  formerly  supposed  that  chlor-iodide  of  zinc  and 
iodine  with  sulphuric  acid  would  always  produce  a  yellow  color 
in  lignified  cell-walls ;  but  it  was  afterwards  found  out  that 
though  this  was  generally  the  case  there  were  a  few  excep- 
tions to  the  rule.  Wiesner29  was  the  first  to  point  out  that 
aniline  sulphate  was  a  reagent  which  in  an  acid  solution  (see  p. 
301)  could  be  used  as  a  test  of  wood  substance  in  every 
kind  of  vegetable  tissue.  Already  Runge  and  Schapringer 
had  microscopically  demonstrated  that  wood  treated  with  this 
substance  assumed  an  intense  yellow  color. 

.  Later  Burgerstein  subjected  the  action  of  aniline  sulphate 
upon  lignin  to  a  very  exact  investigation  and  found  Wiesner's 
views  confirmed  in  all  points.  That  aniline  sulphate  (Wiesner's 
reagent)  is  a  positive  reagent  upon  lignin  follows  from  this, 
that  wherever  this  substance  is  chemically  traceable  in  any 
tissue  it  shows  the  yellow  color,  but  in  all  tissue  from  which 
the  lignin  has  been  withdrawn,  by  powerful  oxidizing  agencies 
such  as  chromic  acid  and  Schultze's  mixture,  the  reagent  leaves 
no  color.30 

Method*1  Put  the  tissue  to  be  tested  in  a  drop  of  distilled 
water  and  let  a  drop  of  the  concentrated  solution  flow  in  from 
the  edge.  In  tissue  full  of  sap  the  reagent  may  be  used 
without  water.  Potassium,  sodium,  and  ammonia  destroy  the 
yellow  color,  but  acid  restores  it  again.  The  color  is  a  pure 
gold  yellow,  yet  the  shade  will  depend  upon  the  quantity 
used. 

Burgerstein  has  investigated  the  different  systems  of  tissue 
and  their  lignification,  by  means  of  aniline  sulphate,  and  has 
arrived  thereby  at  the  following  principal  results. 

Among  thalophytes,  only  certain  lichen  tissue  shows  a  slight 
lignitication.  The  tissue  of  algse  and  fungi  is  never  lignified. 
In  the  vascular  plants  all  the  tissue  systems  are  partially  ligni- 
fied (epidermal  tissue,  tissue  of  the  vascular  bundle,  and  funda- 
mental tissue) . 

29  Wiesnev  in  Karsten's  Botan.  Unters.,  Bd.  I,  p.  120. 

so  Vesque  (Comptes  rendus  de  Paris,  t.  LXX:,  p.  498)  criticises  the  aniline  sulphate, 
since  it  colors  other  membranes  not  Hgnified  yellow.  (  ??;  Hohnel  came  to  the  contrary  con- 
elusion  (Sitzungsber.  d.  K.  Acad.  Wien,  Bd.  LXX VI,  1  Abth.,  p.  528.) 

31  Burgerstein  in  Sitzungsber.  der  K.  Acad.  Wien,  Bd.  LXX,  1  Abth.,  p.  349. 


LIGNIN  AND  ANILINE  SULPHATE.  335 

A.  Epidermal  Tissue.*2     Epidermis,  according  to  Schacht 
and   Dippel,    is   never   lignified.      Burgerstein   confirms   this. 
He  found  this  tissue  lignified  only  in  the  seed  wings  of  Pinus 
and  Abies.    The  cuticle  as  well  as  the  membranes  of  the  stomata 
cells  are  never  lignified.     Hairs  sometimes  are  and  sometimes 
not.     The  collenchyma  tissue  which  supports  the  epidermis  is 
never  lignitied  (Dippel  asserts  the  contrary).33 

B.  Tissue  of  the  Vascular  Bundles.^    The  vessels  in  the 
xylem  are,  with  few  exceptions,  always  lignified  (slightly  so  in 
the  submerged  parts  of  water  plants  and   in  very  sappy  laud 
plants).     Wood-cells  are  always  lignified,  both  in  the  thicken- 
ing cell-walls  and  in  the  middle  lamella,  us  all  naturalists  ad- 
mit.35   The  tertiary  membrane,  "innermost  shell,"  is,  according 
to  Sauio,86. usually  lignified,  but  according  to  Sachs,  Schacht 
and   Dippel  it  is   not.     Burgerstein  agrees  with  Sanio.     The 
wood  parenchyma  is  as   Sanio37  has  already  shown   always  lig- 
nified.    The  bast  cells  are,  according   to  Sachs    and  Schacht, 
sometimes    lignified    and   sometimes   not.     Burgerstein  distin- 
guished :     a,  bast  cells  lignitied   uniformly  in  all  the  layers  of 
the  membranes  with  the  exception  of  the  middle  lamella,  which 
appears  always  to  be  the  most   lignitied    (fully  lignified  bast 
cells)  ;  6,  bast  cells  in  which  the  primary  and  older  secondary 
layers  are   becoming    lignified,    while    the  younger  secondary 
and  tertiary  layers  remain  unlignified   (partially  lignified  bast 
cells)  ;  c,  bast  layers  whose  whole  substance  is  unlignified.   The 
lignitied  bast  cells  are  of  most  frequent  occurrence.     The  sieve 
tubes  are  not  lignified.      The  vascular  bundle  layer  is  always 
more  or  less  lignified. 

C.  Fundamental  Tissue.38     The    pith  cells  are  for  the  most 
part  lignified,  especially  those  lying  next  the  vascular  bundles, 
likewise  the  cells  of  the  medullary  rays.     The  parenchymatous 
fundamental  tissue  is  mostly  not  lignified,  the  leaf  parenchyma 
never.     Sclerenchyma  cells  are  always  lignified. 

32  Bnrgerstein,  L  c.,  p.  344,  ff. 

as  Dippel,  Mikroskop.,  Bd.  II,  p.  155. 

»*  Burgerstein,  1.  c.,  p.  «UU?  ff. 

as  Sanio  in  Pringsheim's  Jahrb.,  Bd.  IX,  pp.  50-126. 

as  Sanio  in  Prings.  Jnhrb.,  I.  c.,  Bot.  Zeit.,  I860,  p.  202. 

a-  S.'iiiio  in  Bot.  Zeitg.,  18«3,  p.  98. 

38  Burgenstein,  1.  c.,  p.  350,  ff. 


336  THE  MICROSCOPE  IN  BOTANY. 

The  lignifying  process  begins  very  early  and  advances  very 
rapi  lly  forward.  First  the  vessels  lignify,  then  the  wood  cells, 
and  the  wood  parenchyma,  very  soon  thereafter  the  bast  cells, 
and  relatively  later  lignification  begins  in  the  pith. 


C.     Behavior  of  Lignin  towards  PJdoroglucin. 

Literature.  Hohnel,  Histochem.  Unters,  iiber  d.  Xylophilin 
u.  d.  Coniferin.  I,  Ueber  d.  Xylophilin  (Sitzungsber.  d.  K. 
Acad.  d.  Wiss.  Wien,  Bd.  LXXVI,  1.  Abth.,  1877,  pp.  663- 
698).— Hohnel,  Ueber  den  Kork,  etc.  (id.,  p.  528).— Wies- 
ner,  Note  iiber  das  Verhalten  des  Phloroglucins  uud  einiger 
verwandter  Korper  auf  verholzte  Zellmembranen  (id.,  Bd. 
LXXVII,  1.  Abth.,  1878,  pp.  60-66).  — Singer,  Beitrage  z. 
naheren  Kenntniss  der  Holzsubstanz  undcler  verholzten  Gewebe 
(id.,  Bd.  LXXXV,  1.  Abth.,  1882,  pp.  345-360).— Poulsen, 
I.  c.,  p.  34  (Translation,  p.  46,  f.).39 

This  reagent  was  discovered  by  Wiesner.  In  an  aqueous  or 
alcoholic  solution  of  even  no  more  than  1  per  cent,  with  the 
addition  of  muriatic  acid,  it  stains  lignin  an  intense  red  violet 
color. 

Method.  Put  the  section  to  be  examined  under  a  cover-glass 
and  add  a  drop  of  the  aqueous  or  alcoholic  reagent  according  to 
circumstances,  and  no  color  will  be  produced.  Then  at  the 
edge  of  the  cover-glass  put  a  drop  of  concentrated  or  somewhat 
dilute  muriatic  acid,  and  directly  a  very  delicate  violet  color  will 
begin  to  enter  the  lignified  tissue,  which  becomes  more  and  more 
intense  till  the  whole  shows  a  uniform,  beautiful  violet  red  color. 
By  reversing  the  process  and  adding  the  acid  first  and  the  rea- 
gent afterwards  the  result  is  the  same ;  the  muriatic  acid  does 
not  color  the  tissue  noticeably.  Vary  the  experiment  by  putting 
a  drop  of  the  reagent  on  a  moist  section  and  evaporating  it  al- 
most all  away  and  then  adding  the  acid  and  the  reaction  takes 
place  almost  instantaneously. 

According  to  v.  Hohnel  we  operate  with  the  extract  of  cherry 

89  References  to  the  literature  belonging  to  this  subject  may  be  found  in  the  works  of 
Borne  of  the  older  writers.  There  is  a  list  of  the  related  literature  in  v.  Hohnel,  I.  c.,  Bd. 
LXXVI,  1  Abth.,  pp.  693-698,  besides  which  see  Weiss  und  Wiesner,  id.,  Bd.  XL,  p.  276. 


LIGNIN  AND  PHLOROGLUCIX.  337 

wood  mentioned  on  p.  303,  in  a  similar  manner.  Add  to  the  fresh 
section  a  little  quantity  of  the  fluid  letting  it  mostly  evaporate 
and  then  add  the  acid.  v.  Hohnel  found  that  in  a  transverse 
section  of  the  stem  of  the  Anthericum  liliago,  by  this  treatment 
the  epidermis  and  the  soft  parenchyma  lying  directly  beneath 
the  young  pith  ancl  the  soft  bast  cells  remained  perfectly  color- 
less. The  vessels  and  the  middle  lamella  of  the  woody  tissue 
became  dark  violet ;  the  more  imperfectly  lignified  thickening 
layers  of  the  wood  cells  and  the  elements  of  the  sclereuchyma 
sheath  bright  violet.40 

A  transverse  section  through  the  stem  of  Rumex  obtusifolius 
treated  with  an  alcoholic  solution  of  phloroglucin  and  concen- 
trated muriatic  acid  showed  the  following.  The  epidermis  and 
the  strongly  developed  collenchyma  layer  lying  beneath  as  well 
as  the  next  following  thin  bark  parenchyma  remained  perfectly 
colorless.  In  the  vascular  bundles  all  the  woody  parts  inclusive 
of  the  less  numerous  wide  vessels  were  colored  a  dark  red 
violet.  At  the  beginning  of  the  reaction  the  color  began  to 
become  distinct  in  the  middle  lamella  first ;  afterwards  it  ex- 
tended itself  uniformly  through  the  whole  of  the  lignified  walls. 
The  outer  layers  of  the  pith  are  likewise  strongly  lignified 
corresponding  to  their  red  violet  coloring.  Towards  the  center 
of  the  section  the  pith  cells  become  gradually  a  brighter  violet 
and  at  the  center  remained  quite  colorless  since  they  are  not 
in  the  least  lignified. 

If  the  above  described  section  were  put  in  distilled  water  the 
violet  red  color  would  be  changed  to  a  brick  red  which  would 
grow  paler  by  degrees  till  at  last  the  section  would  be  quite 
colorless.  It  behaves  the  same  way  in  alcohol  or  ether.  The 
water  dissolves  the  phloroglucin  because  the  latter  forms  no 
chemical  combination  with  the  wood  substance,  it  being  only 
mechanically  absorbed  and  intercalated  in  the  cell  walls.  A 
section  was  carefully  wrashed  in  water  for  a  long  time  (five 
hours)  and  in  spite  of  that  took  a  faint  violet  color  when  muriatic 
acid  was  added,  showing  that  there  still  remained  a  small  quan- 
tity of  the  phloroglucin  to  produce  the  reaction.  But  if  one 
removes  the  adhering  acid  from  the  section  by  passing  it  through 

«  V.  Hohnel,  I.  c.,  Bd.  LXXXI,  1  Abth.,  p.  686. 
22 


338  THE  MICROSCOPE  IN  BOTANY. 

water  and  then  adds  ammonia  the  color  will  change  instantly  to 
yellow  and  cloudy  orange ;  the  parts  which  before  were  tinted 
the  intense  violet  will  now  show  the  deep  shade  of  yellow.  If 
now  the  alkali  be  washed  out  and  acid  again  applied  the  violet 
color  will  be  restored  and  of  the  same  intensity  as  before. 
Ammonia  (sodium  or  potassium  lye,  a  basic  salt)  produces  the 
decolorization  of  the  ^phloroglucin  stain.  Muriatic  acid  (sul- 
phuric acid,  nitric  acid,  acid  salts)  restores  the  coloring  again 
(see  also  v.  Hohnel,  I.  c.). 

D.     Behavior  of  Lignin   towards   Indol. 

Literature.  Niggl,  Das  Indol  ein  Reagenz  auf  verholzte 
Zellmembranen.  Mikrochernische  Untcrsuch.  (Flora,  1881,  pp. 
545-559,  561-566  ;  also  separate  as  a  dissertation.  Regensburg, 
1881,  22  pages). —  Singer,  Beitrage  zur  naheren  Kenntniss  der 
Holzsubstanz  uiid  der  verholzten  Gewebe  (Sitzungsber.  d.  K. 
Acad.  d.  Wiss.  Wien,  Bd.  LXXXV,  1  Abth.,  1882,  pp. 
346-360). 

Indol,  according  to  the  recently  published  investigations  of 
Niggl,  produces  a  stain  quite  like  that  of  the  phloroglucin  re- 
action.41 By  means  of  an  acid  it  colors  lignified  membrane 
from  a  cherry  red  to  red  violet. 

Method.  The  section  to  be  examined  is  put  on  the  slide  with 
a  drop  of  the  aqueous  solution  of  indol  and  a  cover-glass  laid 
over  it.  Then  by  means  of  a  piece  of  blotting  paper  draw  out 
a  part  of  the  solution  and  let  flow  in  1  to  2  parts  of  the  dilute 
sulphuric  acid  mentioned  on  p.  304,  whereupon  the  reaction 
immediately  takes  place.  The  specimen  thus  prepared  keeps 
its  beautiful  color  a  long  time.  If  concentrated  acid  be  used, 
or  the  superfluous  quantity  bo  not  drawn  off,  the  color  of  the 
lignified  membranes  in  a  few  weeks  will  be  changed  to  brown- 
red.  To  prevent  this  let  the  acid  work  for  an  hour  or  two  and 
then  draw  it  out  with  filter  paper  and  replace  with  glycerine. 

Not  only  does  the  phloroglucin  and  indol  reactions  show  the 

41  Still  several  other  substmces  have  been  proposed  for  the  same  purpose  in  recent 
times,  asPyrol  (Niggl),  Orcin  (Lippmann),  Resorcin  (Molisch,  Wiesner),  Pyrogalin  (Wies- 
ner),  Hydrochinon  (Niggl).  1  have  not  tested  the  working  of  all  these  substances, 
on  a'ccount  of  the  scarcity  of  some  of  them.  On  the  contrary,  I  have  carefully  followed 
out  Niggl's  statements  regarding  indol  and  find  them  to  be  correct  in  all  cases. 


LIGNIN  AND  INDOL.  339 

greatest  agreement  in  respect  to  coloring,  but  also  as  I  have 
found  in  the  behavior  of  the  colored  membranes  towards  bases 
and  acids.  Wash  out  the  acid  from  the  indol-stained  section 
and  substitute  ammonia  and  the  violet  color  wilt  disappear  and 
in  its  place  will  appear  a  yellow  to  ochre  colored  tint.  If  this 
again  be  washed  out  and  sulphuric  acid  be  added  the  violet  color 
will  be  restored.  To  distinguish  it  from  phloroglucin  it  is  to  be 
mentioned  that  the  indol  stain  does  not,  like  that  of  phloroglu- 
cin, disappear  by  prolonged  treatment  with  water.  Sections 
stained  with  indol  retain  their  color  with  undiminished  intensity 
when  they  have  lain  in  water  for  twenty- four  hours.  This 
makes  the  indol  reaction  preferable  to  that  of  phloroglucin. 

Just  as  Btirgerstein  tested  the  effect  of  aniline  sulphate  on 
the  different  systems  of  tissue,  Niggl  has  studied  that  of  indol. 
We  give  in  the  following  a  brief  resume  of  his  results.  For 
the  purpose  of  a  more  easy  comparison  with  those  of  Burger- 
stein  on  p.  335  they  are  presented  in  the  same  consecutive  order. 

THALLOPHYTES. 

In  algse  there  appears  to  be  no  lignification  except  in  the 
stout,  warty,  thickened  membranes  of  some  Cosmarium  species. 
In  the  greater  part  of  the  fungi  no  kind  of  coloring  appears. 
The  exceptions  are  Polyporus  fomentarius  (a  glimmer  of  red), 
Ochrolechia  pallescens  and  Trametes  suaveolens  (distinct  red). 
The  thallus  of  the  lichens  behaves  variously.42 

VASCULAR  PLANTS. 

A.  Epidermal  Tissues.  The  epidermis  is  not  colored  by 
indol  and  sulphuric  acid.  The  exception  to  this  is  the  epi- 
dermal cells  of  the  leaves  of  Cinnamomum  Culilawan,  Cycas 
revoluta  suidflexuosa,  and  of  the  needles  of  several  Coniferce. 
The  cuticle  is  generally  not  lignified.  The  young  sprouts 
of  ^Esculus  hippocastanum,  Acer  pseudoplatanus  and  Hippuris 
vulgaris  are  an  exception  to  this.  The  cuticle  consists  of 
numerous  scales,  and  these  often  show  a  difference  of  behavior, 
the  inner  ones  being  sometimes  reddened  by  indol.  The  mem- 

41  Niggl,  1.  c.,  separatbdr.,  p.  5,  /. 


340  THE  MICROSCOPE  IN  BOTANY. 

brane  of  the  hairs  is  as  frequently  lignified  as  not.  The  sto- 
mata  cells  of  the  Ooniferce  and  the  Cycadeaz  are  frequently 
colored  red  by  indol.  Collenchyma  tissue  is  not  lignified.  The 
collenchyma  of  the  stem  and  leaves  of  Sapindus  laurifolius 
is  an  exception  to  this  rule. 

B.  Tissue  of  the  Vascular  Bundles.     The   vessels,   are  al- 
ways lignified,  the  wood  cells  also,  middle  lamella  and  thick- 
ening layer  always;  tertiary  membrane  (inner  shell)  remains 
uncolored   in   Astragalus,    Caragana,  Robinia   and     Cytisus. 
The  cells  of  the  wood  parenchyma   are    always  lignified   and 
indeed  all  three  layers ;  in  its  younger  state  the  innermost  is 
not  fully  colored.     The  bast  cells  show  considerable  variation. 
Niggl  observed,  as  did  Burgerstein,  bast  cells  perfectly  lignified 
and  others  totally  lacking  in  that;  frequently,  however,  they 
were   partially  lignified.     The   outer   layer,    especially  in  the 
younger   stages,  is  commonly  reddened,  while   the  inner  one 
remains  uncolored.     Later,  the  middle  part  shows  the  reaction 
on  the  lignin.     The  sieve  tubes  are  not  lignifijed.     The  sheath 
of  the  vascular  bundles  is  always  at  least  partially  lignified.43 

C.  Fundamental  Tissue.      Pith  cells  are  commonly  ligni- 
fied ;  the  cells  of  the  medullary  rays  always,  except  in  Aristo- 
lochia  sipho.     The  hypoderm  is  sometimes  liguified,  but  rarely 
the  leaf  parenchyma  (Cycas  revoluta,   C.  flexuosa).     In   the 
sclerenchyma   cells   the    lignification  can   always    be    demon- 
strated. 

The  lignifying  process  begins  earliest  in  the  vessels. 

E.     Behavior  of  Lignin  towards  Phenol -muriatic  Acid. 

Literature.  Tiemann  und  Haarmann,  Ueber  d.  Coniferin 
seine  Unwandlung  in  das  aromat.  Princip  der  Vanille  (Ber. 
Deutsch.  Chem.  Gesellsch.,  Bd.  VII,  1874,  p.  608,/*.)—  Tangl, 
Vorlauf.  Mitth.  liber  die  Verbreitung  des  Coniferin  (Flora, 
1874,  p.  239,  /".).— Rud.  Mtiller,  Ueber  Coniferin  (I.  c.,  p. 
399). —  v.  Hohnel,  Ueber  den  Kork  und  verkorkte  Gewebe 
iiberhaupt  (Sitzungsber.  d.  K.  Acad.  d.  Wiss.  Wien,  Bd. 
LXXVII,  1  Abth.,  1877,  p.  700,  ff.).—v.  Hohnel,  Histochem. 

*8  Particulars  in  Niggl,  I.  c.,  pp.  12-14. 


LIGNIN  AND   PHENOL-MURIATIC  ACID.  341 

Unters.  iiber  d.  Xylophilinu.  d.  Coniferin.  II,  Ueber  d.  Conif- 
eriu  (id.  p.  699,  ff.). — Singer,  Beitrag.  z.  naheren  Kennt- 
niss  d.  Holzsubstanz  und  verholzten  Gewebe  (id.  Bd. 
LXXXV,  1  Abth.,  1882,  p.  347,  ff.) 

It  has  long  been  known  to  the  chemist  that  a  pine  shaving 
passed  through  carbolic  and  muriatic  acid  becomes  blue.  After- 
wards it  was  proved  by  Tiemann  and  Haarmann  that  this  coloring 
depended  upon  a  substance  existing  in  wood  discovered  by  Th. 
Hartig  and  called  coniferin.  Tangl  showed  about  the  same 
time  that  a  like  reaction  took  place  not  only  in  coniferous 
wood,  but  also  in  Sambucus  niyra,  Populus  bahamifera,  Frax- 
inus  excelsior  and  Vitis  vinifera.  v.  Hohnel  afterwards  ad- 
vanced the  hypothesis  that  coniferin  is  an  element  of  all  wood 
tissue  and  that  therefore  the  carbolo-rnuriatic  reaction  would 
serve  as  a  test  for  wood  tissue  in  general.  Singer  confirmed 
the  views  of  v.  Hohnel  in  a  recently  published  investigation. 
While  it  was  at  first  supposed  that  the  reaction  took  place  by 
first  moistening  with  carbolic  acid  and  then  with  muriatic  acid, 
v.  Hohnel  first  observed  that  the  action  of  direct  sunlight  was 

o 

also  necessary. 

Method  (v.  Hohnel).44  Use  the  phenol-muriatic  acid  de- 
scribed on  p.  302.  With  the  perfectly  clear  solution  we  may 
obtain  very  clean  and  beautiful  preparations.  The  section 
should  not  be  too  thin,  and  being  moistened  the  least  possible 
with  the  reagent  it  should  be  put  under  a  cover-glass  and  set  in 
the  direct  sunlight.  An  exposure  of  from  one-half  to  one 
minute  will  be  sufficient.  A  short  time  after  this  the  section 
will  have  an  intense  color.  If  the  exposure  is  for  a  longer 
time  the  strength  and  vividness  of  the  color  slowly  fade  and 
it  becomes  a  sea-green  or  a  yellow-green.  The  beautiful  green 
color  is  characteristic  of  the  really  lignified  membranes.  All  cells 
which  would  be  colored  blue  with  chlor-iodide  of  zinc,  the  epi- 
dermis, bark  which  is  destitute  of  wood  and  cellulose,  remain 
uncolored  or  colored  yellowish  by  the  muriatic  acid  in  the  re- 
agent.45 The  section  must  be  immediately  examined  because 

4«  V.  H6hnel,.Z.  c.,  Bd.  LXXVI,  p.  700,  ff. 

45  Muriatic  acid  colors  all  wood  tissues  and  also  some  others  a  more  or  less  intense 
yellow.  The  color  is,  however,  mostly  very  weak,  the  addition  of  water  readily  destroy, 
ing  it. 


342  THE  MICROSCOPE  IN  BOTANY. 

the  green  color  is  not  durable.  In  lack  of  direct  sunlight,  con- 
centrated artificial  light  may  be  substituted,  though  with  poorer 
results.  Very  beautiful  preparations  are  made  from  the  aerial 
roots  of  orchids,  stems  of  monocotyledonous  plants,  woods  of 
Evonymus,  ^Esculus  and  the  Coniferce. 

According  to  Tommaso  and  Donato  Tommasi46  the  "conifer!  11 
reagent"  is  more  distinctly  effective  if  the  section  to  be  treated  is 
first  moistened  with  a  mixture  of  carbolic  acid  and  potassium 
chlorate,  and  then  with  the  muriatic  acid.  By  this  means  the 
blue  stain  comes  out  in  diffused  light,  more  rapidly  and  more 
intensely,  and  the  preparation  will  not  lose  its  color  in  a  day. 


For  the  purpose  of  determining  the  relative  sensitiveness  of 
lignin  reactions,  Singer47  has  experimented  with  solutions  of 
like  concentration  of  phloroglucin,  indol,  pyrol,  aniline  sul- 
phate, resorciu,  paratoluidin,  pyrogalic  acid,  etc.,  by  observing 
the  effect  of  each  upon  tissue  uniformly  lignified,  and  has 
arrived  at  these  results  :  "  That  with  a  1  per  cent  solution  of 
phloroglucin,  indol  and  pyrol,  we  are  able  to  produce  vivid 
colors,  quite  uniform  in  their  intensity.  But  also  much  weaker 
dilutions  of  the  last  named  reagents  carried  were  able  to 
produce  coloring,  and  in  a  0.001  per  cent  concentration  the 
limit  of  the  effectiveness  of  phloroglucin  was  reached  (see  also 
p.  303),  while  the  indol  reduced  to  a  0.0007  per  cent  dilution 
colored  coniferous  wood,  particularly  after  several  hours.  Py- 
rol is  effective  only  when  used  in  a  stronger  dilution. 

Indol  is  thus  the  most  sensitive  reagent  which  we  possess  for 
testing  lignification.  But  it  does  not  prove  to  be  the  most  use- 
ful;  for,  not  to  mention  its  great  costliness  (1  g.  costing  70 
M.)  [in  America  $45],  it  will  not  keep  well  and  requires  the 
greatest  caution  in  working  with  sulphuric  acid  which  in  a 
concentrated  form  destroys  all  vegetable  tissue. 

In  consideration  of  this  and  in  respect  to  the  fact  that  pyrol 
is  very  difficult  to  make  and  changes  its  nature  after  a  few 

46  Tommaso  and  Donato  Tommasi,  Ueber  d.  Fichtenholzreaction  zur  Entdeckung  des 
Phenols  im  Urin.  (Ber.  Deutsch.  chem.  Gesellsch.,  1881,  p.  1834,  JT.)  —  Cf.  auch  Singer,  I.  c., 
p.  353,  ff. 

«  Singer,  1.  c.,  p.  358. 


INTER-CELLULAR   SUBSTANCE.  343 

hours,  we  must  give  the  preference  to  phlorogluciu  in  combina- 
tion with  muriatic  acid  over  all  other  lignin  reagents." 

After  my  experiments  with  aniline  sulphate,  phloroglucin  and 
inclol,  I  have  to  add  to  this  that  I  agree  with  Singer  in  respect 
to  the  sensitiveness  of  indol ;  but  that  it  appears  to  me  as  if 
the  indol  deserves  the  preference  over  the  phloroglucin.  Prep- 
arations stained  with  it  seem  to  keep  far  better  than  when 
treated  with  phloroglucin,  if  one  will  carefully  wash  out  the 
acid  with  distilled  water  and  preserve  in  glycerine.  The  use  of 
sulphuric  acid  of  a  dilution  of  one  to  four  sufficiently  insures 
the  specimen  against  harm.  And,  finally,  in  respect  to  the  de- 
composition of  indol  I  must  remark  that  I  have  an  aqueous 
solution  which  now  for  more  than  seven  months  has  perfectly 
preserved  its  efficiency  and  also  its  pungent  smell. 

4.     MIDDLE  LAMELLA,  Intercellular  Substance. 

Literature.  Mohl,  Verm.  Schr.,  p.  314  ff.,  etc. —  Mohl,  Die 
veget.  Zelle,  p.  196. — Wigand,  Intercellularsubstanz  und  Cu- 
ticula,  Brschwg.,  1850.  Schacht,  Lehrb.  d.  Anat.  u.  Phys.  d. 
Gew.,1856,  Bd.  I,  p.  108.  —  Sanio,  Ueber  Intercell.  im  Holz 
(Bot.Zeitg.,  1860,  p.  208-213). — Vogl, Ueber  d.  Intercellulars. 
u.  die  Milchsaftgef.  in  d.  Wurzel  des  gem.  Lowenzahns  (Sit- 
zungsber.  der  K.  Acad.  d.  Wiss.  Wien,  Bd.  XLYIII,  2  Abth., 
1863,  pp.  668-690) — Wiesner,  Unters.  liber  d.  Auftretenv.Pec- 
tinkorpern  in  den  Geweben  d.  Runkelriibe  (id.  Bd.  L,  2 
Abth.,  1865,  pp.  442-453) — Hofmeister,  Lehre  v.  d.  Pflanzen- 
zelle,  1867,  sec.  31. — Wiesner,  Einl.  in  d.  techn.  Mikroskopie, 
Wien,  1867,  pp.  62,  244,  246,  etc. — Dippel,  Die  Intercellulars. 
und  deren  Entstehung.  Rotterd.,  1867. — Dippel,  Mikroskop, 
Bd.  II,  p.  99  ff.— Sachs,  Lehrb.,  p.  72.— Dippel,  D.  neuere 
Theorieiiberd.  feinereStructurd.Zellhiille,etc.  (Schr.  d.  Senck- 
enbergischen  Gesellsch.,  Bd.  X,  XI,  1875-78,  p.  41  ff.).— 
Solla,  Beitr.  z.  naheren  Kenntn.  der  chem.  und  physikal.  Bes- 
chaffenh.  der  Intercellulars.  (Oesterr.  bot.  Zeitschr.  1879,  pp. 
341-353).— v.  Hohnel,  Notiz  iiber  d.  Mittellamelle  der  Holz- 
elemente,  etc.  (Bot.  Zeitg,  1880,  p.  450^".) — See  also,  in  part, 
the  writings  cited  on  pages  319  and  333. 


344  THE  MICROSCOPE  IN  BOTANY. 

Under  the  conception  of  the  middle  lamella  we  are  to  under- 
stand the  homogeneous,  sharply-defined  partition  lying  between 
two  contiguous  cells  and  which  appears  to  be  transformed 
from  the  primary  cellulose  membrane  by  chemical  metamor- 
phosis48 and  then  often  assumes  certain  conditions  of  solubility. 
It  is  always  recognizable  in  wood  tissue  by  being  easily  seen. 
With  many  anatomists  the  term  "  intercellular  substance  "  is  used 
as  synonymous  with  "middle  lamella."  But  Sachs  and  Wiesner 
distinguish  between  the  two  expressions  and  apply  the  former 
term  to  the  jelly  or  pectin-like  substance  which  sometimes  lies 
between  the  cells  and  which  is  formed  by  a  chemical  metamor- 
phosis in  which  more  or  less  of  the  cell  wall  becomes  homoge- 
neous (endosperm  of  Ceratonia  siliqua,  the  tissue  of  the 
Fnci,  etc.). 

The  views  of  botanists  concerning  the  production  and  chem- 
ical nature  of  the  middle  lamella  or  intercellular  substance  have 
greatly  differed.  Schacht49  supposed  the  intercellular  substance 
to  be  a  binding  cement  between  the  cells  distinct  from  the  cellu- 
lose. According  to  Dippel50  the  middle  lamella  (which  he 
calls  the  primary  cellulose  covering)  is  not  homogeneous,  but 
consists  of  two  corresponding  layers  of  cellulose  and  of  one  of 
intercellular  substance  lying  between,  which  latter  proceeds 
from  the  cambium  walling  of  the  tissue  cells,  which  is  formed 
rom  a  combination  essentially  different  from  cellulose  but  iso- 
meric  with  it,  tind,  indeed,  of  the  daughter  and  not  the  mother 
cells  of  the  tissue,  which  latter,  as  soon  as  they  have  fulfilled 
their  function,  are  dissolved  and  reabsorbcd.  That  combination 
essentially  favors  the  dissolving  of  the  contiguous  coverings  of 
the  cells  and  allows,  in  consequence,  transformations  which  are 
not  peculiar  to  cellulose. 

According  to  Wigand,51  the  intercellular  substance  arises  from 
the  intimate  commingling  of  the  primary  cell  walls,  which 
view  was  also  advanced  by  Sanio52  who  added  that  the  intercel- 

48  A  strict  distinction  must  be  made  between  the  primary  membrane  and  the  middle  la- 
mella, which  in  some  botanical  hand  books  is  not  expressly  done. 
*9  Lehrb.  d.  Anat.  u.  Physiol.  d.  Gew.,  Bd.  I,  p.  129. 
50  Dippel,  Mikroskop,  Bd.  II,  p.  105 /. 

61  Wigand,  Botan.  Unters.,  p.  79. 

62  Sanio  in  Bot.  Zeitg.,  1860,  p.  210  ff. 


INTER-CELLULAR   SUBSTANCE.  345 

lular  substance  was  altogether  or  partially  lignified,  and,  indeed, 
this  lignification  takes  place  sometimes  before  that  of  the  secon- 
dary cell  layer.  It  colors  yellow  with  chlor-iodide  of  zinc,  but 
if  the  intercalated  wood  substance  is  removed,  by  boiling  in 
potash  the  reactio'n  of  chlor-iodide  of  zinc  will  be  that  of  cellu- 
lose. The  views  of  the  two  last-named  naturalists  are  current 
to-day. 

The  more  recent  investi  orations  of  Sol  la53  teach  that  the  inter- 
cellular substance  or  middle  lamella,  in  the  course  of  the  devel- 
opment of  the  tissue,  enters  into  different  chemical  as  well  as 
physical  transformations.  It  is  molecularly  distinct  from  the 
adjoining  layers  of  cell  wall.  The  first  foundation  of  the  inter- 
cellular substance  is  either  pure  cellulose  (cambium)  or  (at  the 
point  of  the  stem),  a  substance  in  which  cellulose  is  afterwards 
traceable  in  the  young  permanent  tissue.  The  intercellular  sub- 
stance of  the  young  permanent  tissue  consists,  as  a  rule,  of 
cellulose.  In  perfectly  formed  permanent  tissue  cellulose  is  but 
rarely  traceable  (in  many  kinds  of  bast)  ;  commonly  it  enters 
into  many  metamorphoses  and  then  exhibits  toward  the  reagent 
a  very  different  behavior.  These  metamorphoses  lead  finally 
sometimes  to  the  complete  separation  of  connected  cells. 

Reactions.  Of  the  iodine  reagents,54  iodine  and  sulphuric 
acid,  or  chlor-iodide  of  zinc,  produce  in  most  cases  a  yellow 
coloring  of  the  middle  lamella :  after  previously  boiling  it  in 
potash  lye  these  reagents  give  a  blue  or  violet  tinted  color 
(cellulose  reaction)  ,K  This  latter  color,  however,  always  appears 
at  the  outset  when  the  middle  lamella  consists  of  cellulose.  Par- 
tially lignified  middle  lamella  shows  a  corresponding  mixture  of 
colors  between  yellow  and  violet.  Boiling  nitric  acid  in  combina- 
tion with  ammonia  often  gives  a  strong  yellow  color  to  the  middle 
lamella  (Solla,  v.  Hohnel).  Phloroglucin  and  indol  behave 
toward  the  middle  lamella  very  much  as  toward  the  lignified 
thickening  layers  of  the  cells.  When  the  reaction  takes  place 
gradually  the  middle  lamella  colors  before  and  more  intensely 
than  the  adjoining  layers.  (See  p.  337.) 

53  Solia  in  Oesterr.  Bot.  Zeitschr.  1879,  pp.  341-353. 
««  Sanio,  I.  c.,  Taf.  VI,  Figs.  10-12, 15. 
65  Sanio,  I.e.,  Taf.  VI,  Fig.  16. 


346  THE  MICROSCOPE  IN  BOTANY. 

Dissolving  reagents,  on  the  contrary,  behave  very  differently 
toward  the  intercellular  substance  and  its  solubility  is  not 
proportionate  to  its  age.56  The  rule  is  that  cuprammonia, 
concentrated  sulphuric  acid  and  dilute  chromic  acid  ap- 
plied cold  will  not  dissolve  it  (see  p.  317),  while  concentrated 
chromic  acid  will  do  so  with  difficulty  and  Schultze's  maceration 
mixture,  easily.57  The  intercellular  substance  of  very  delicate 
tissue  will  sometimes  be  dissolved  partially  or  altogether  by  the 
action  of  boiling  water  (for  example  in  the  parenchyma  of 
the  beet  root,  Wiesner).  Acetic  acid  dissolves  the  intercellular 
substance  of  the  potato  after  a  long  time,58  tartaric  and  oxalic 
acid  very  slowly  (Wiesner,  Solla).  Potash  lye,  nitric  acid 
and  muriatic  acid  dissolve  it  very  rapidly  (potato,  pith  of 
jSambucus).  The  middle  lamella  of  wood  is  most  rapidly 
dissolved  in  boiling  nitro-muriatic  acid  and  strong  chromic 
acid.  Potash  lye  slowly  dissolves  the  intercellular  substance 
of  certain  bast  fibres  as  in  the  parenchyma  of  the  beet  root 
(Wiesner) . 

Intercellular  substance  may,  in  certain  cases,  undergo  a  pec- 
tose  metamorphosis.  Mulder59  and  Kabsch60  first  showed  that 
pectose  occurs  in  many  cell  walls  ;  the  latter  also  showed  that 
in  the  boundary  layer  of  the  cells  it  is  most  intimately  mingled 
with  cellulose  and  appears  as  intercellular  substance.  Yogi61 
further  found  that  in  the  root  of  the  dandelion  the  intercellular 
substance  is  produced  by  the  transformation  of  cellulose  into 
pectose.  Wiesner  studied  the  appearance  of  the  pectose  bodies 
in  the  beet  root  and  found,  in  agreement  with  Kabsch  and  Vogl, 
that  the  intercellular  substance  is  the  seat  of  the  pectose  which 
is  principally  a  product  of  the  transformation  of  the  outer  layer 
of  the  mother  cell,  but  that  not  only  parenchyma  tissue,  but 
also  cambium,  vascular  and  wood  cells  and  likewise  peridermal 

£6  Wiesner  in  Sitzungsber.  d.  K.  Acad.  d,  Wiss.  Wien,  Bd.  LXH,  1  Abth.  p.  201. 

w  According  to  Wiesner  (Einleit.  in  d.  techn.  Mikrosp.  p.  47)  chromic  acid  will  always 
dissolve  the  intercellular  substance  (see  also  Wiesner  in  Proceed.  Imperial  Acad.  of  Science, 
Vienna,  Vol.  LXII,  part  1,  p.  200).  According  to  H.  MUller,  the  intercellular  substance  of 
wood  is  dissolved  by  bromine  water  (official  report  of  the  Vienna  World's  Exposition,  1873, 
Brunswick,  1877,  Vol.  Ill,  1  part,  2nd  half,  p.  27^). 

68  Solla,  1.  c.,  p.  344. 

59  Mulder,  Physiol.  Chem.,  p.  514. 

e° Kabsch  in  Pringsheim's  Jahrb.,  Bd.  Ill,  p.  3G7. 

«i  Vogl,  Sitzungsber.  d.  K.  Acad.  Wiss.  Wien,  Bd.  XL VIII,  2  Abth.  1863,  p.  668  ff. 


CORKY   CELLULOSE.  347 

cells  may  contain  pectose.62  This  pectose  metamorphosis  may 
lead  in  certain  cases  to  the  formation  of  jelly  and  to  the  loosening 
or  separating  of  the  individual  cells.  It  maybe  mentioned  also 
that  pectic  acid  salts  are  detected  in  many  plants ;  according  to 
Fremy,  calcium  pectate  is,  in  many  of  the  tissues,  the  cementing 
medium  of  the  cells.  According  to  Maudet  it  is  an  element  of 
the  pith  of  Aralia  papyrifera.  According  to  Gireaud  pectic 
acid  is  found  in  large  quantities  in  gum  tragacanth.63 

The  presence  of  pectose  substances  is  demonstrated  by  the 
cell  walls  swelling  in  boiling  water  and  potash  lye,  and  dissolv- 
ing in  the  latter.  According  to  Poulsen64  cuprammonia  will  pre- 
cipitate in  tissue  containing  pectose  a  copper  pectinate,  which  in 
thin  sections  will  still  remain  after  the  entire  disintegration  of  the 
rest  part  of  the  membrane. 

The  methods  of  investigating  intercellular  substances  with 
reagents  have  been  sufficiently  given  in  treating  of  lignin. 
Should  we  wish  to  dissolve  these  substances  by  heating  the  re- 
agent, the  operation  may  be  conducted  with  a  watch-glass  which 
should  not  be  heated  over  a  free  flame,  but,  following  the  process 
of  Sanio,65  should  be  placed  upon  a  thin  plate  of  iron  and  this 
heated  till  the  contents  of  the  glass  boil. 

5.    CORKY  CELLULOSE,  SUBERIN. 
(Including  Cutin,  Pollenin.) 

Literature.  Kroker,  De  plantar,  epidemide  observ.  Yratisl., 
1833.— Mohl,  Unters.  iiber  d.  Entwickl.,  des  Korkes  u.  d. 
Borke  auf  der  Rinde  der  baumart.  Dikotyl.  (Verm.  Schr.,  pp. 
212-232  ;  auch  Diss.  aus  d.  Jahre,  1836).— Mohl,  Unters.  tiber 
d.  Lenticellen  (id.,  pp.  233-245;  auch  Diss.  vom.  Jahre, 
1836). — Mohl,  Ueber  d.  Cuticula  der  Gewachse  (id.,  pp.  260- 
368;  auch  Linnaea,  1842). — Fritsche,  Ueber  den  Pollen, 
Petersbg.,  1837. — Nageli,  Entwicklungsgesch.,  d.  Pollens,  etc., 
Zurich,  1842.— Cohn,  De  Cuticula.  Yratisl.,  1850.— Schacht, 

«2  Wiesner,  ibid,  Bd.  L,  2  Abth,  1864,  p.  450. 
63  Husemann,  Pflanzenstoffe,  Bd.  1. 1882,  p.  186. 
"Poulsen,  Botan.  Mikrochem.,  p.  57,  Trans,  pp.  15,  91. 
e5  Sanio  in  Bot.  Zeit.,  1860,  p.  211,  Anm. 


348  THE   MICROSCOPE  IN  BOTANY. 

D.  Pflanzenzelle,  p.  239. — Hanstein,  Ueber  d.  Ban  u.  d.  Ent- 
wickl.  d.  Baumrinde,  Berlin,  1853. — Fremy,  Recherches  chim. 
sur  la  cuticule  (Comptes  rendus  de  Paris,  t.  XLVIII,  1859,  p. 
667,  ff.). — Sanio,  Ueber  d.  Bau.  u.  die  Entwickl.  des  Korkes 
(Pringsheim's  Jahrb.,  Bd.  11,1860,  pp.  39-108).— Schacht, 
Ueber  d.  Bau  einiger  Pollenkorner  (id.,  pp.  109-159). — Pol- 
lender,  die  Chroms.,  ein  Losungsmittel  fur  Pollenin  u.  Cutin 
(Bot.  Zeitg.,  1862,  p.  405). — Faivre,  Sur  les  plaies  d'ecorce 
par  incis.  annul,  et  sur  leurs  effets,  etc.,  Paris,  1864. — Fllicki- 
ger,  Lehrb.  d.  Pharmakogn.  d.  Pflanzenreiches,  Berlin,  1867, 
p.  336. — De  Bary,  Ueber  d.  "VVachsuberziige  der  Epidermis 
(Bot.  Zeitg.,  1871,  p.  128,  ff.)—  Pfitzer,  Beitr.  z.  Kenntn. 
d.  Hautgewebe  d.  Pfl.  (Pringsheim's  Jahrb.,  Bd.  VII,  p.  532, 
ff.9  Bd.  VIII,  p.  73,  ff.)—  Hegelmaier,  Ueber  d.  Bau  u.  die 
Entwickl.  einiger  Cuticulargebilde  (ed.,Bd.  IX,  p.  286,  ff.). 
— Haberlandt,  Ueber  d.  Nachweisung  der  Cellulose  ira  Kork- 
gewebe  (Oesterr.  bot.  Zeitschr.,  1874,  pp.  229-234).— Miiller, 
E-.,  Die  Rinde  unserer  Laubholzer.  Bresl.,  1875. — Tschistia- 
koff,  Ueber  d.  Entwicklungsgesch.  des  Pollens  v.  Epilobium 
angusti folium  (Prings.  Jahrb.,  Bd.  X,  p.  7-45). ^-v.  Hohuel, 
Ueber  den  Kork  und  verkorkte  Gewebe  iiberhaupt  (Sitz- 
ungsber.  d.  K.  Acad.  d.  Wiss.  Wien,  Bd.  LXXVI,  1  Abth., 
1877,  p.  507-562;  cf.  auch  Bot.  Zeitg.,  1877,  p.  783,  ff.). 
— v.  Hohnel,  Ueber  d.  Cuticula  (Oesterr.  Bot.  Zeitg., -1878, 
No.  3,  u.  4). — Niggl,  D.  Indol,  eiu  Reagenz  auf  verholzte 
Zellmembranen,  p.  9,  f. 

Corky  or  cuticularized  cellulose  is  distributed  through  the 
well-known  cork  layer,  which  is  often  solid,  free  from  inter- 
cellular spaces,  mostly  consisting  of  not  very  much  thickened 
cell  layers  and  their  derivatives,  also  in  the  endodermis,  and  in 
that  fine  continuous  coating  which  is  drawn  over  the  outer  walls 
of  the  epidermis  cells,  and  finally  in  the  outer  inclosing  sheath  of 
pollen  grains  and  many  spores.  According  to  De  Bary's66  and 
especially  v.  HohnePs67  investigations  the  suberization  does 
not  seize  upon  all  portions  of  the  cork  cell  region,  but  it  is 

««De  Bary  in  Bot.  Zeitg.,  1871,  p.  128,  ff. 

«  v.  Hohnel  in  Sitzungsber.  d.  K.  Acad.  Wiss.  Wien,  Bd.  LXXVI,  1  Abth.,  p.  507. 


CORKY  CELLULOSE.  349 

limited  to  certain  definite,  often  sharply  marked,  zones.  Ac- 
cording to  v.  Hohnel,  almost  eyery  cork  cell  wall  (exclusive  of 
many  young  cork  cells  of  the  Coniferaz) ,  which  belongs  to  two 
neighboring  cells,  consists  of  the  following  five  lamella:  (1)  of 
a  middle  strongly  lignified  plate,  which  is  not  distinguishable 
from  the  middle  lamella,  or  is  only  partially  lignified ;  (2)  of 
two  suberized  layers  which  lie  on  the  two  sides  of  this  one ; 
(3)  of  two  cellulose  layers  which  lie  next  to  the  two  last  and 
also  to  the  cell  space,  and  which  are  more  or  less  strongly 
lignified. 

The  cork  substance  forming  the  suberin  layer  is  as  little 
known  in  respect  to  its  chemical  nature  as  is  lignin.  Accord- 
ing to  Mitscherlich,  Dopping  and  others,  cork  substance  is  dis- 
tinguished by  containing  1.50  per  cent  to  2.3  per  cent  of 
nitrogen,  while  according  to  v.  Hohnel  there  are  no  grounds 
for  supposing  it  to  contain  nitrogen,  since  albuminous  sub- 
stances have  never  anywhere  been  detected  in  suberin.  But 
suberin  contains  from  73  to  74  per  cent  of  carbon  and  10  per 
cent  of  hydrogen  (by  which  it  follows  that  it  must  contain 
from  16  to  17  per  cent  of  oxygen).  It  is  insoluble  in  boiling 
alcohol  and  stands  in  its  chemical  as  in  its  physical  nature  be- 
tween wax  and  cellulose.  De  Bary68  has  shown  that  frequently, 
perhaps  always,  in  the  formation  of  cuticle,  a  molecular  interca- 
lation of  wax  takes  place.  A  very  characteristic  physical 
quality  of  the  cork  lamella  is  that  it  is  almost  entirely  imper- 
meable by  diosmosis,  as  Sanio,69  by  a  series  of  very  striking 
experiments,  has  already  proved. 

As  in  other  modifications  of  cellulose,  so  in  the  suberine 
lamella,  pure  cellulose  may  often  be  detected  when  the  "in- 
crusting  substance"  has  been  removed  by  a  process  already 
described  on  pages  318  and  331.  The  first  who  directed  at- 
tention to  this  characteristic  were  Mohl70  and  Hofmeister71. 
The  former  noticed  the  cellulose  in  the  cork  of  a  flask  after 

«8De  Bary  in  Dot.  Zeitg.,  1871,  p.  593,  ff. 

69  Sanio  in  Pringsheim's  Jahrb.,  Bd.  II,  p.  54,  /.—See  also  De  Bary,  I.e.;  Hanstein  in 
Bot.  Zeitg.,  1868,  pp.  70S,  748;  Behrens  in  Flora,  1879,  p.  374,  /. 
TO  H.  v.  Mohl  in  Bot.  Zeitg.,  1847,  p.  497. 
"Hofmeister  in  Ber.  d.  K.  Sachs  Gesellsch.d  .  Wiss.  Leipzig, Bd.  X  (4858),  p.  21. 


850  THE  MICROSCOPE  IN  BOTANY. 

maceration  in  potash  lye.  The  latter  showed  that  the  lami- 
nated cuticular  layer  of  epidermis  cells  of  Hoja  carnosa  gave  a 
very  distinct  blue  coloring  with  iodine  reagent  when  it  had  been 
excluded  from  the  air  and  treated  for  two  or  three  weeks  with 
concentrated  potash  lye.  The  same  reaction  appears  in  the 
cuticle  of  the  leaves  of  Orchio  morio  after  previous  treatment 
with  concentrated  sulphuric  acid.  "The  principal  reasons  for 
including  cuticle  among  those  membranes  which  are  essentially 
different  from  cellulose  falls  to  the  ground  with  this  proof."72 
According  to  De  Bary73  the  cuticular  substance  of  the  leaves  of 
Klopstockia  is  very  easily  destroyed  by  a  warm  ten  per  cent 
solution  of  potash  whereof  the  pure  cellulose  walls  remained 
behind.  Haberlandt,  who  has  made  the  most  searching  inves- 
tigation of  the  occurrence  of  cellulose  in  cork  found  that  the 
test  may  be  made  by  maceration  in  chromic  acid,  and  Schultze's 
mixture,  but  preferably  by  boiling  the  section  to  be  tested 
in  potassium  chlorate  and  nitric  acid,  and  then  before  the  section 
quite  falls  apart  treat  it  for  some  moments  with  boiling  potash 
lye.  Then  after  washing  it  out  with  water  the  membranes  of 
the  separated  tissue  will  be  colored  an  intense  blue  by  cblor- 
iodide  of  zinc  and  be  dissolved  by  cuprammonia*74 

The  suberized  parts  of  the  membrane  may  be  easily  recog- 
nized as  such  by  some  characteristic  reactions.  Like  the 
middle  lamella  it  is  perfectly  insoluble  in  cuprammonia  and  con- 
centrated sulphuric  acid.  The  latter  often  colors  the  cuticular- 
ized  extine  of  many  pollen  grains  usually  a  beautiful  rose-red,75 
seldom  yellow.  Acetic  acid  causes  the  extine  of  many  spores  of 
ferns  to  swell.76 

Concentrated  chromic  acid  itself  will  not  dissolve  suberin,  or 
if  at  all  with  the  greatest  difficulty  ;  v.  Hohnel  uses  this  therefore 
as  a  test  of  suberin  (chromic  acid  reaction)  :77  use  a  pure  quite 
concentrated  solution.  It  causes  the  suberized  membrane  to 
stand  out  clear  and  distinct  while  the  rest  part  of  the  tissue  first 

72  See  also  v.  Mohl,  Vermischte  Schr.,  p.  263. 

73  De  Bary,  1.  c.  p.  578. 

74  Haberlandt,  in  Oesterr.  Bot.  Zeitsch.,  1874,  p.  232,  /. 

76  Schacht  in  Priugsheim's  Jahrb.,  Bd.II,  p.  134,  etc.  Taf.  XVIII,  Figs.  10, 11, 14,  15,  31, 32. 

76  Fischer  v.  Waldheim  in  Priugsheim's  Jahrb.,  Bd.  IV,  p.  375. 

77  v.  Hohnel,  I.  ti.t  p.  526. 


CORKY  CELLULOSE.  351 

becomes  gradually  more  indistinct  and  then  entirely  disappears. 
Suberized  membrane  is  dissolved  by  chromic  acid,  as  previously 
noted  only  with  much  difficulty  but  after  treatment  by  it  for 
eight  or  ten  hours  it  becomes  transparent.  Very  strongly  sub- 
erized  membrane  holds  out,  however,  for  a  week,  but  finally 
becomes  transparent.  Wash  out  the  acid  and  again  it  becomes 
dark  and  distinct  as  before.  Cuticularized  membranes  have 
the  same  characteristics. 

Iodine  with  sulphuric  acid  as  well  as  chlor-iodide  of  zinc  col- 
ors suberized  cellulose  yellow  or  brown  or  deep  brown  itself. 
According  to  Mohl78  if  the  cuticle  is  impregnated  with  iodine  it 
is  colored  a  deep  yellow  or  brown.  If  the  specimen  is  treated 
with  sulphuric  acid  the  cuticle  is  dissolved  off  and  can  be  seen 
very  well.  Sometimes  by  this  process  it  becomes  a  still  darker 
brown.79  The  extine  of  the  pollen  grain  colors  with  iodine  and 
sulphuric  acid  quite  the  same  way.83  So  also  do  many  spores, 
as  for  example,  fern  spores. 

Indol  with  sulphuric  acid  (see  p.  339)  leaves  suberized  cells, 
as  in  the  cuticle,  quite  uucolored,81  at  least  the  suberized  lamellae 
never  show  any  coloring,  while  in  the  walls  of  old  cork  cells  a 
red  coloring  is  noticeable  after  this  treatment.  But,  as  can  be 
shown  in  very  thin  sections,  only  the  middle  lamella  is  stained. 
Young  cork  cells  show  no  reddening.  Concentrated  potash  lye 
produces  no  noticeable  change  in  cork  tissue  except  a  very  faint 
yellow  coloring.  But  by  holding  the  slide  over  a  small  flame, 
slowly  heating  it  but  not  quite  to  the  boiling  point,  the  color 
becomes  darker,  the  membrane  itself  a  little  swollen  and  at 
least  a  definite  layer  of  the  wall  assumes  a  granular  appear- 
ance (pure  cellulose  membrane  only  swells  but  for  the  rest 
maintains  a  smooth  surface) .  By  boiling  the  granulization  be- 
comes more  pronounced  and  in  most  cases  the  granular  and 

78  v.  Mohl,  Verm.  Schr.,  p.  261. —  For  particulars  concerning  the  coloring  of  cuticle  see 
pp.  260-207,  and  compare  with  illustrations  in  Tables  IX  and  X. 

7»  According  to  Hofmeister  (Ber.  Sachs,  Gesselsch.,  Leip.Bd.  X,  p.  21),  the  cuticle  of  the 
seed  of  Linum  usitatissimum  behaves  in  a  characteristic  manner.  By  treatment  with 
iodine  and  dilute  sulphuric  acid  it  is  colored  blue  the  blue  bordering  on  the  black.  Add- 
ing concentrated  sulphuric  acid  changes  the  color  to  yellow.  Washing  out  the  acid  with 
water  restores  the  blue  color. 

*»  Schacht,  1.  c.,  p.  103-168. 

si  Xiggl,  1.  c.,  p.  9. 


352  THE  MICROSCOPE  IN  BOTANY. 

variegated  substances  protrude  from  the  membrane.  If  now 
the  section  be  washed  with  water  the  granular  masses  will  be 
for  the  most  part  destroyed.  It  now  becomes  evident  that  every 
cell  wall  of  cork,  however  thin  it  may  be,  consists  of  three  mem- 
branous lamellae  (see  p.  348),  a  middle  one  in  common  and  two 
which  belong  to  the  adjoining  cells,  which  lamellae  are  often 
separated  from  each  other  by  a  wide  space  between.  These 
spaces  were  originally  filled  with  this  granular  mass.  Suber- 
ized  cells  treated  with  a  cold  concentrated  solution  of  potash 
take  on  a  yellow  color  while  all  other  cell  membranes  re- 
main almost  or  altogether  uncolored.  By  heating,  the  yellow 
color  of  the  first  becomes  more  intense,  and  that  of  the 
latter  when  there  is  any,  more  pale  (potassium  reaction,  v. 
HOhnel).82 

By  boiling  the  section  under  investigation  in  Schultze's  mix- 
ture (see  p.  163)  the  suberized  membrane  becomes  very  in- 
distinct, while  the  rest,  even  strongly  lignified  tissue  itself, 
gradually  becomes  transparent.  Cuticle  and  cuticularized  tissue 
behave  in  the  same  way.  Warmed  under  a  cover-glass  a  violent 
development  of  gas  takes  place  and  then  the  suberized  tissue 
alone  remains.  ,  Now  wash  out  the  Schultze's  mixture  and  add 
alcohol  and  then  ether  and  the  whole  becomes  hyaline.  But  if 
the  heating  is  carried  still  further  the  membranes  suddenly  swell 
and  melt  together  into  masses  which  finally  become  quite  glob- 
ular, consist  of  eerie  acid,  and  are  soluble  in  hot  alcohol,  ether, 
benzole,  chloroform  and  dilute  potash  lye.  To  recognize 
slightly  suberized  tissue,  lay  the  section  a  short  time  in  cold 
Schultze's  mixture  wash  out  and  add  potash  lye.  By  the  former 
the  suberized  membrane  becomes  somewhat  more  distinct  and 
by  the  latter  it  takes  an  ocher  yellow  color  and  becomes  crumbly. 
If  this  does  not  immediately  happen  slightly  warming  commonly 
helps  it  on.  At  the  same  time  the  potash  lye  produces  a  fur- 
ther clarification  of  the  suberized  membrane  (eerie  acid  re- 
action, v.  Hohnel).83 

82  V.  Hohnel,  pp.  522-524. 

83  v.  Hohnel,  I.  c.,  pp.  524-526. 


FUNGUS  CELLULOSE.  353 


6.    PUNQUS  CELLUT,OSE. 

Literature.  Schacht,  Die  Pflanzenzelle,  p.  13. —  Dippel,  D. 
Mikroskop,  Bd.  II,  p.  7,  /. — De  Bary,  Morphology  der  Pilze, 
Flechtenund  Myxomycetin  (Hofmeister,  de  Bary,  Sachs,  Hand- 
buch  d.  Physiol.  Botan.,  Bd.  II,  p.  7,  /).— Poulsen,  Bot. 
Mikrochemi,  p.  51  (Trans,  p.  79). — Richter,  Beitrage  z.  Gen- 
aueren  Kennta.  derchem.  Beschaffeuheit  d.  Zellmembranen  bei 
den  Pilzen  (Sitzungsber.  d.  K.  Acad.  d.  Wiss.  Wien,  Bd. 
LXXXIII,  1  Abth.,  1881,  pp.  494-510).  [Ber.  Deutsche  Bot. 
Gesellsch.  1(1883),  pp.  288-308  (1  pi.),  Jour.  Roy.  Mic. 
Soc.,  Vol.  Ill,  part  V,  p.  676.] 

The  membranous  substance  which  forms  the  walls  of  the 
hyphae  of  fungi  and  lichens,  fungus  cellulose,  was,  until  the 
recent  investigations  of  Schacht,  Dippel  and  De  Bary,  looked 
upon  as  an  entirely  different  substance  to  cellulose,  since  it  had 
not  been  possible  before  that  by  any  known  medium  of  macer- 
ation to  remove  the  incrusting  substance  so  as  to  produce  by 
iodine  reagents  the  cellulose  reaction.  According  to  Schacht  and 
De  Bary  the  fungus  walls  themselves  were  not  colored  blue  with 
iodine  and  sulphuric  acid  after  boiling  in  potash  lye,  nor  indeed 
after  treatment  with  Schultze's  mixture  or  chromic  acid.  For  the 
rest  it  was  already  known  by  De  Bary  and  others  that  many 
plant  membranes  become  blue  by  iodine  or  chlor-iodide  of  zinc 
without  any  other  previous  treatment.  In  Mucor  it  was  demon- 
strated that  in  the  young  state  the  cell  walls  were  colored  blue 
with  iodine  and  sulphuric  acid,  but  in  the  older  stages  they  re- 
mained colorless. 

In  later  investigations  Richter  opposed  the  view  that  fungus 
cellulose  is  essentially  different  from  true  cellulose.  He  suc- 
ceeded in  removing  the  incrusting  substance  from  the  fungus 
Polyporus,  after  long  and  numerous  treatments  of  the  fungus 
tissue  with  water,  hot  potash  lye,  acetic  acid,  alcohol,  ether 
and  again  water,  and  then  in  producing  the  cellulose  reaction 
with  chlor-iodide  of  zinc.  The  same  results  were  produced 
with  fungi  and  lichens  by  macerating  the  tissue  continuously 

23 


354  THE  MICROSCOPE  IN  BOTANY. 

fur  two  or  three  weeks  in  potash  lye  frequently  changing  the 
macerating  liquid.84  The  fungus  membrane  thus  purified 
was  colored  a  rose  red  to  a  violet  by  chlor-iodide  of  zinc  ;  it  also 
appeared  to  be  dissolved  by  cuprammonia  but  this  could  not 
be  definitely  verified.  Richter85  did  not  succeed  in  demon- 
strating any  lignification  of  these  membranes  by  means  of 
aniline  sulphate  or  phlorogliicin,  not  even  in  the  lichen  itself.86 
On  the  other  hand,  he  demonstrated  the  most  distinct  suberiza- 
tion  in  Daedaka  quercina  by  means  of  the  eerie  acid  reac- 
tion (see  p.  351). 

The  purification  of  the  membranes  of  fungus  cells  so  as  to 
get  the  cellulose  reaction  is  generally  accomplished  only  with  the 
greatest  difficulty,  and  the  utmost  patience  is  necessary. 
Richter87  found  by  his  studies  that  the  fungus  cellulose  is  but 
common  cellulose  with  an  admixture  of  foreign  matter,  perhaps 
albuminous  substances ;  that  a  fungus  cellulose  in  the  sense  of 
De  Bary  does  not  exist. 

In  its  natural  state  fungus  cellulose  is  distinguished  by  its 
extraordinary  resistance  to  the  different  reagents.  It  is  perfectly 
insoluble  in  cuprammonia,  can  be  scarcely  touched  by  cold 
potash  lye,  muiiatic  acid  and  Schultze's  mixture.  Concen- 
trated sulphuric  acid  destroys  it  only  with  the  greatest  difficulty. 
On  the  contrary,  it  should  be  distinctly  stated  that  many  fungus 
membranes  are  soluble  in  muriatic  acid. 

[Pringsheim  has  further  investigated  the  peculiar  granules 
long  since  observed  by  him  in  the  fertilizing  tubes  and  oogonia 
of  the  Saprolegniese  and  described  by  Zopf  as  amoebae.  They 
are  found  in  the  fertilizing  tubes  at  all  ages.  While  young  they 
are  flat  disk-shaped  or  polyhedral  plates  with  rounded  corners 
composed  of  a  dense  homogeneous  substance.  They  vary  greatly 
in  size  and  form.  They  gradually  become  stratified  and  at  last 
as  regularly  and  completely  as  starch  grains.  They  are  abun- 
dant also  in  the  oogonia  and  a  few  grains  occur  in  other  parts 
of  the  plant.] 

si  Of  lichens  those  must  be  selected  which  have  the  least  possible  amount  of  lichenin  in 
them  since  this  gives  the  same  reaction  as  cellulose  (Richter,  1.  c.,  p.  503.  See  above  p. 
270). 

85  Richtev,  1.  c.,  p.  505,  f. 

so  On  the  contrary,  see  Burgerstein  above  p.  2S3,  and  Xiggl  above,  p.  287. 

«  Richter,  I.  c..  p.  510. 


FUNGUS  CELLULOSE.  355 

[The  structure,  mode  of  development  and  chemical  properties 
of  these  substances  show  that  they  are  neither  organs  of  re- 
production nor  independent  parasitic  organisms  but  are  a  special 
modification  of  the  cell  contents.  The  stratification  indicates  a 
close  resemblance  to  other  bodies  of  this  character.  They  are, 
however,  not  colored  blue  by  iodine  nor  do  they  take  any  other 
color  but  that  of  the  iodine  itself.  They  are  completely  in- 
soluble in  all  ordinary  solvents  of  oils  and  resins,  even  in  abso- 
lute alcohol  and  ether.  Nitric  acid  either  with  or  without 
ammonia  or  potash  produces  no  effect  on  them  nor  does  Millon's 
reagent.  They  have  no  power  of  taking  up  coloring  substances 
except  under  special  circumstances.  Caustic  alkalies  cold 
produce  no  visible  effect  on  these  bodies  and  very  little  change 
is  effected  by  dilute  or  concentrated  nitric  or  hydrochloric  acid 
at  common  temperature.  In  moderately  concentrated  sulphuric 
acid  they  dissolve  rapidly  and  completely,  at  the  ordinary  tem- 
perature, as  also  in  solutions  of  zinc  chloride  when  not  too 
dilute.  They  do  not  dissolve  in  cuprammonia  even  after  long 
treatment.] 

[These  reactions  show  that  the  bodies  in  question  belong 
,  neither  to  the  proteinaceous  cell  contents,  nor  to  the  series  of  oils 
and  resins,  but  that  they  are  composed  of  a  substance  closely  al- 
lied to  cellulose  which  has  been  separated  from  the  protoplasm  in 
a  granular  form.  It  is  perhaps  identical  with  so-called  "fungus 
cellulose"  and  with  the  "fibrose"  of  Fremy,  and  Pringsheim  pro- 
poses for  it  the  term  "cellulin."  Its  special  chemical  characteris- 
tic is  its  remarkable  solubility  in  dilute  sulphuric  acid,  and  in  an 
aqueous  solution  of  zinc  chloride.] 

The  stratification  of  the  cellulin  grains  is  concentric  around 
a  nucleus  of  denser  substance.  They  grow,  however,  to  a  con- 
siderable size  before  any  stratification  is  evident.  Compound 
grains  are  not  uncommon.  A  common  mode  of  multiplication 
is  by  a  kind  of  budding  not  dissimilar  to  that  of  torula. 

[When  the  oospores  are  formed  out  of  the  protoplasmic  con- 
tents of  the  organism,  an  unused  residue  remains  behind,  which 
is  the  substance  out  of  which  the  cellulin  grains  are  subsequent- 
ly developed.  This  substance  is  morphologically  identical  with 


356 


THE  MICROSCOPE  IN  BOTANY. 


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STARCH,  AMYLUM.  357 

the  "periplasm"  of  the  Peronosporece  out  of  which  the  exospores 
of  the  oospore  is  formed.  Quoted  from  Journal  Royal  Micro- 
scopical Society,  I.  c.  A.  B.  H.] 


Having  finished  our  presentation  of  the  microscopical  exami- 
nation of  cellulose  we  present  the  various  reactions  in  a  tabular 
form  for  greater  convenience  in  using  them.  It  will  be  very 
easy  to  identify  by  means  of  this  table  the  different  kinds  of 
cellulose  which  are  subjected  to  examination.  Muculent,  as  well 
as  fungus  cellulose  has  been  excluded  from  this  schedule  since 
by  their  peculiar  outward  appearance,  or  the  manner  of  their 
occurrence,  they  are  immediately  recognized  as  such.  In  the 
species  of  cellulose  cited  we  have  had  in  mind  only  the  pure 
types,  and  in  those  cases  where  it  has  been  transformed  into 
another  or  mingled  in  the  membranous  layer  with  another  (as, 
for  example,  in  the  partial  lignification  of  suberiu  lamellae)  the 
reader  will  have  to  consult  the  foregoing  chapter. 


II.     STARCH,  AMYLUM. 

Literature.  Fritsche,  Ueber  das  Amylum  (PoggendorPs  Ann. 
Bd.  XXXII,  1834,  p.  129,  ff.)—  Payen,  Compos.  Elem.  de 
PAmidon  de  diverges  Plantes,  etc.  (Annales  de  Chim.  et  de 
Phys.,  t.  LXV,  1837,  p.  225  ff.)—  Nageli,  Blaschenfg.  Gebilde 
im  Inhalte  d.  Pflzelle,  7,  Starkeblaschen,  Starkekoruer  (Zeitsch. 
f.  wiss.  Bot.  v.  Schieiden  und  Nageli,  Heft  3,  4,  p.  117,  ff.)— 
Mohl,  D.  veget.  Zelle  (Wagner's  Haudworterb.  Bd.  IV,  1851, 
p.  207.) — Maschke  in  Erdmanu's  Archiv,  f.  prakt.  Chem.,  1852, 
2,  p.  400. — Walpers,  Beitrag.  z.  Kenntn.  d.  Amylums  (Flora, 
1852,  p.  689  ff,  705  ff.)— Hartig,  Ueber  die  Ban  des  Starke- 
mehls  (Bot.  Zeitg.,  1855,  No.  52  —  Nachtragdazu  ebendaselbst, 
1856,  p.  349  ff.) — Melseus,  L'lustitut,  1857,  p.  161. — Cramer, 
Verh.  d.  Kupferoxydammoniaks,  z.  Zellmernbran,  Starke  Inu- 
lin,  etc.,  Zurich,  1857. — Nageli,  D.  Starkekorner,  Zurich,  1858. 
— Nageli  u.  Cramer,  Pflauzeuphysiol.  Unters.  II  (1858),  p.  113 


358  THE  MICROSCOPE  IN  BOTANY. 

ff.  181  /".—  Hartig,  Entwicklungsgeschich.  d.  Pflkcims,  1858, 
pp.  88,  155. — Mohl,  Unters.  d.  Pflanzengewebes  mit  Hilfe  des 
polarisirteu Lichtes  (Bot.  Zeitg.,  1858,  p.  1, j^.) — Sachs,  Ueber 
eiuige  neue  Reactionsmethodin  (Sitzungsber.  d.  K.  Acad.  d. 
Wiss.  Wien,  Bd.  XXXVI,  1859,  p.  5,  ff.)—  Mohl,  Ueber  d. 
vorgeblichen  Gehalt  d.  Starkekorner  an  Cellulose  (Bot.  Zeitg. 
1859,  p.  225  jf.  233 /*.)— Sachs,  Ueber  d.  Auftreten  d.  Starke 
bei  d.  Keimung  olhaltiger  Samen  (id.  p.  177  ff.,  185  ff.) — 
Sachs,  Mikrochem.  Unters.  (Flora,  1862,  p.  299/".)— Nageli, 
Ueber  d.  Reactionen  v.  Jod  auf  Starkekorner  u.  Zellmembranen 
(Ber.  d.  Bayer.  Acad.,  1862,  1863,  Bd.  I,  p.  161  jf.  483/.)- 
Nageli,  Ueber  d.  Chem.  Verschiedenh.  d.  Starkekorner  (id. 
1863,  Bd.  II,  p.  272,  ff.) — Sachs,  Ueber  d.  Entstehung  der 
Starke  in  den  Blattern  (Monatshefte  d.  Annalend.  preuss.  Land- 
wirthsch.,  1863) — Sachs,  Ueber  d.  Stoffe  welche  das  Material 
z.  Wachsthum  derZellhaute  liefern  (Pringsheim's  Jahrb.,Bd.  Ill 
1863,  p.  183, /".)— Mulder,  Physiol.  Chem.,  p.  217. — Sachs, 
Handb.  d.  Experimentalphysiol.  d.  Pflanz.  p.  412,  ff. — Hof- 
meister,  Handb.  d.  physiol.  Bot.,Bd.  I,  p.  387, /.— Dippel,  D. 
Mikroskop,  Bd.  II,  p.  24. — Nageli  u.  Schwendener,  D.  Mikro- 
skop,  p.  512, /.—Sachs,  Lehrb.  d.  Bot.,  p.  59  ff.—W.  Nageli, 
Bietr.  z.  naheren  Kenntniss  der  Starkegruppe,  1874. 

Starch  or  Amylum  (C6H10O5  or,  according  to  W.  Nageli, 
C36H62O3l)  is  isomeric  with  cellulose  and  is  almost  universally 
distributed  as  solid  contents  of  plant  cells.  It  occurs  in  all 
the  phanerogams  but  has  not  yet  been  observed  in  the  fungi 
and  some  families  of  algae.  It  is  the  first  visible  product  of  as- 
similation by  the  chlorophyll  grains,  and  is  formed  within  theni.* 
At  certain  times  in  the  life  of  the  plant  it  is  changed  into  the 
form  of  a  fluid  isomeric  carbo-hydiate  (glycose)  and  circulates 

*  J.  Boehm  (in  liotan.  Zeitg.  XLI  (1883)  pp.  33-38,  49-54)  contests  the  ordinary  view  that 
starch  formed  in  the  chlorophyll  grains  is  a  direct  result  of  the  decomposition  of  carbon 
dioxide.  He  believes  it  to  be  in  many  cases  formed  from  sugar  or  other  organic  substances 
which  have  found  their  way  into  the  chlorophyll  grains.  Leaves  and  pieces  of  the  stem  of 
the  scarlet  runner  containing  no  starch  were  exposed  to  the  action  of  a  solution  of  sugar 
when  they  were  found,  after  24  hours,  to  contain  an  abundance  of  starch,  the  quantity  de- 
pending on  the  concentration  of  the  sugar  solution.  This  author  thinks  therefore  that  sugar 
and  not  starch  is  the  first  demonstrable  product  of  the  decomposition  of  carbon  dioxide. 
Quoted  from  Jour.  Roy.  Mic.  Soc.,  Vol.  Ill,  No.  Ill,  pp.  388-90.  A.  B.  H. 


STARCH,  AMYLUM. 


359 


to  other  parts  of  the  plant  to  he  again  transformed  into  starch 
in  the  parenchyma,  the  medullary  rays  of  the  stem,  rhizomes, 
tubers,  bulbs  and  seeds.  There,  after  having  been  inactive  for 
a  long  time,  as  a  reserve  substance,  it  will  furnish  rich  material 
for  the  building  up  of  new  organs  in  a  new  period  of  vegetable 
growth  after  first  being  changed  into  sugar  and  dextrine.  The 
vessels,  as  it  appears,  are  always  free  from  starch. 


TIG.  132. 

It  is  very  often  easy  to  recognize  starch  as  such  by  its  outward 
appearance.  It  forms  roundish,  transparent,  colorless,  elliptical, 
egg-shaped,  irregular  grains,  simple  or  compound,  of  from  0.001 
mm.  to  0.15  mm.  in  diameter.  The  starch  grain  is  laminated, 
the  laminae  lying  about  a  mostly  excentrically  placed  nucleus 
which,  in  the  young  grain,  is  filled  with  aqueous  substances 
and  in  the  old  grain  with  air.  The  layers  are  optically  distinct 


360  THE  MICROSCOPE  IN  BOTANY. 

by  reason  of  their  'differing  contents  of  water  (Fig.  121,  p. 
263).  When  the  lamination  is  not  easily  seen  the  addition  of 
dilute  chromic  acid  will  make  it  more  distinct.88 

Alcohol,  on  the  contrary,  makes  the  lamination  less  dis- 
tinct. Starch  grains  disperse  the  light  into  two  rays  of 
unlike  velocity  which  polarize  at  right  angles  to  each  other. 
Therefore  they  appear  bright  in  the  polarizing  apparatus 
(see  p.  133)  with  the  prisms  crossed,  bright  with  a  dark 
cross  running  over  them  and  passing  through  the  before 
mentioned  nucleus  (Fig.  132).  Starch  grains  behave  like 
double  refracting  crystals  and  we  assume,  therefore,  that  they 
consist  of  a  mixture  of  anisotropic  substances. 

Of  all  reagents  employed  in  testing  starch,  iodine  solutions 
hold  the  first  place.  Colin  and  Gaultier  de  Claubry  observed 
as  early  as  1814,  that  a  solution  of  iodine  gave  a  blue  coloring 
to  starch.  By  it  the  starch  was  changed  to  an  iodine  amylum. 
The  color  which  iodine  imparts  to  starch  seems  to  depend 
upon  the  kind  and  concentration  of  the  solution  used,  and  will 
be  violet,  indigo-blue,  or  a  deep  dark  blue.  The  color  is  com- 
monly a  full  indigo  blue  ;  a  pure  blue  does  not  occur  (Nageli). 
All  iodine  reagents  produce  this  coloring  by  the  intercalation  of 
iodine  atoms  in  the  starch  grain.  The  reaction  is  so  distinct 
that  starch  granules  of  but  2  to  3  ^  in  diameter  are  certainly 
recognizable  as  such.  The  intensity  of  the  bluing  is  not  propor- 
tional to  the  iodine  contents  of  the  solution.  The  quantity  of 
the  intercalated  iodine  conditions  the  intensity  of  the  color  of 
the  starch  grains  but  it  can  produce  no  distinction  in  the  tone 
of  the  color  (Nageli). 

The  bluing  takes  place  only  when  the  starch  grains  contain 
water  (Mohl).  An  alcoholic  solution  of  iodine  colors  anhy- 
drated  amylum  very  slowly  a  brown  yellow.  If  the  iodine 
alcohol  is  mixed  with  much  water  it  gives  a  brown  or  violet 
tone  (Nageli). 

By  the  use  of  potassium  iodide  of  iodine  the  bluing  takes  place 
very  quickly,  more  slowly  with  iodine  water  and  most  slowly 

88  Dippel,  D.  Mikroskop,  Bd.  I,  p.  276. 


STARCH,  AMYLUM.  361 

of  all  with  iodine  glycerine.  The  last  named  reagent  should  be 
used  in  studying  the  very  gradual  effect  of  iodine.89  According 
to  the  investigations  of  Nageli,90  potato  starch  in  water  colors 
on  the  addition  of  some  iodine  at  first  a  bright  blue,  but  after- 
ward an  intense  indigo  blue.  If  the  iodine  is  removed,  the  color 
gradually  disappears ;  but,  before  it  becomes  quite  colorless,  it 
shows  again  the  bright  violet.  If  the  iodine  reagent  contains 
no  other  substance  than  iodine  (potassium  iodide,  hydriodic 
acid,  etc.),  the  starch  will  retain  its  blue  color  on  dry  ing.  But 
in  other  cases,  if  it  has  taken  in  another  substance  it  will  change 
color  through  violet,  red  and  orange  to  yellow,  in  exact  rela- 
tion to  the  quantity  of  the  absorbed  substance.  Starch  grains 
in  zinc  chloride,  by  the  addition  of  a  splinter  of  iodine,  take 
the  color  with  extraordinary  slowness.  Before  the  grain  swells 
it  becomes  red  violet  or  pale  violet,  and  afterwards  a  pure 
blue.  Concentrated  solution  of  zinc  chloride  like  sulphuric 
acid  disintegrates  the  starch  grains  into  many  small  particles. 
If  the  solution  of  chlor-iodide  of  zinc  is  made  very  dilute  with 
water,  the  starch  grains  will  not  swell  up,  and  will  become 
blue  violet  or  indigo  blue. 

"In  exactly  the  same  treatment  with  iodine  reagents  the  dif- 
ferent parts  of  a  starch  grain  and  the  different  kinds  of  starch 
behave  differently  ;  perhaps  the  one  having  a  somewhat  stronger 
affinity  for  the  iodine  colors  more  rapidly,  and  so  takes  on  a 
somewhat  different  tone  of  color. 

The  particular  grain  of  starch,  or  the  particular  layer  of  a 
starch  grain,  gives  with  iodine  a  different  color  according  to  the 
nature  and  the  quantity  of  the  foreign  substances  which  have 
permeated  it  (water,  acids,  salts,  indifferent  organic  combina- 
tion, etc.),  according  to  whether  these  substances  enter  the 
starch  grains  before  or  after  the  iodine,  and  according  to  whether 
the  iodine  still  keeps  its  original  arrangement,  or  is  already 
making  preparations  to  leave  the  starch. 

The  colors  which  the  iodine  can  produce  in  the  starch  are 
indigo,  violet,  red,  orange  and  yellow.  They  depend  on  a  pecu- 

89  Hartig,  I.  c.;  see  for  the  rest,  Jsageli,  Bayer.  Acad.  1863, 1,  p.  188. 

90  Xageli,  1.  c.,  p.  161  ff. 


3ti2  THE  MICROSCOPE  IN  BOTANY. 

liar  arrangement  of  the  particles  of  iodine  and  are  in  general  noth- 
ing else  than  what  is  recognized  in  the  iodine  itself  in  a  solid, 
liquid  and  gaseous  state. 

Of  the  different  starch  and  iodine  combinations  the  blue 
corresponds  to  the  strongest  affinity,  the  yellow  to  the  weakest. 
When  the  iodine  enters  the  starch  it  always  assumes  that  mole- 
cular arrangement  which  the  greatest  possible  affinity  requires, 
under  the  given  conditions ;  if,  on  the  contrary,  it  is  compelled 
to  leave  the  starch  by  some  other  force  it  previously  changes  its 
molecular  constitution  in  the  manner  that  corresponds  to  the 
weaker  affinity.  The  presence  of  water  always  conditions  the 
color  corresponding  to  the  stronger  absorption  and  intercalation 
of  the  particles  of  iodine,  the  presence  of  some  other  substance 
on  the  contrary  produces  that  color  which  corresponds  to  the 
weaker  affinity."91 

Concerning  the  behavior  of  some  other  substances  towards 
starch  the  following  may  be  mentioned. 

Bromine,  with  water,  colors  starch  grains  a  pure  orange 
yellow. 

Starch  is  insoluble  in  water,  alcohol  and  ether,  insoluble  but 
swelling  in  concentrated  iodine  solutions,  chlor-iodide  of  zinc, 
bromine  solutions,  calcium  chlorate  and  cuprammonia  (the  ad- 
dition of  water  restores  the  grains  to  their  original  volume), 
soluble  in  all,  even  the  very  dilute,  mineral  acids.  The  latter 
finally  change  it  into  combinations  of  the  dextrine  group. 
Cuprammonia  colors  starch  grains  dark  blue.  If  starch  in 
water,  potash  lye,  dilute  mineral  acids  and  certain  organic 
acids  be  warmed  to  about  50°  it  becomes  structureless  and  then 
the  well-known  paste-forming  process  begins. 

According  to  the  investigations  of  Nagcli  the  starch  grain 
consists  of  two  essentially  different  components,  granulose  which 
gives  the  characteristic  iodine  reaction,  and  starch  cellulose. 

If  starch  be  digested  by  a  gentle  heat  with  saliva  fluid,  or 
with  pepsin  (Melsens),  or  treated  with  chromic  acid,  the  gran- 
ulose, that  component  of  the  starch  grain  which  is  blued  with 
iodine  will  be  withdrawn,  and  the  element  which  remains,  the 

91  Nageli,  1.  c.,  pp.  176, 197, 198. 


STARCH,  AMYLUM.  363 

delicate  framework  of  starch  cellulose,  will  not  then  color  blue 
with  iodine  reagents,  but  for  the  most  part  the  same  as  cellulose 
(Xageli).92  Iodine  water,  or  fresh  iodine  alcohol  stains  it  if  at 
all,  a  weak,  pale  copper  red.  After  drying,  potassium  iodide 
of  iodine,  chlor-iodide  of  zinc  and  iodine  alcohol  produce  a 
violet  to  an  indigo  blue  color  (Nageli) .  Micro-chemistry  distin- 
guishes starch  cellulose  from  many  other  kinds  of  characteristic 
cellulose  by  its  easy  solubility  in  potassium  hydroxide  and  chlor- 
iodide  of  zinc  solution.  Starch  cellulose  dissolves  in  cupram- 
monia  (v.  Mohl). 

It  still  remains  to  be  shown  how  starch  is  tested  when  it  is 
enveloped  in  protoplasmic  substances,  or  when  it  is  found  with- 
in the  chlorophyll  grains. 


A.    STAKCH  MINGLED  WITH  PROTEID 
PLASMIC  SUBSTANCES.93 

In  very  young  and  small-celled  parenchyma  tissue  which  is 
developed  from  the  primary  meristem  of  the  vegetation  point, 
starch  is  almost  never  to  be  discovered  by  means  of  iodine 
alcohol  or  potassium  iodide  of  iodine.  If  chlorophyll  is  not  present 
a  thin  section  is  warmed  in  the  strongest  potassium  hydroxide, 
or  left  to  lie  in  it  cold  for  a  considerable  time,  then  washed 
with  water  and  neutralized  with  acetic  acid  and  iodine  alcohol 
much  diluted  with  water  added.  Then  with  strong  magnification 
either  swollen  blue  grains  will  be  seen  in  the  yellow  plasma 
or  a  blue  paste.  In  the  latter  case  it  will  naturally  be  impos- 
sible to  determine  if  the  starch  occurs  here  in  grains  or  in 
solution  perhaps  intimately  united  with  the  nitrogenous  con- 
tents of  the  plasma. 

If  the  plant  tissue  which  has  been  washed  out  contains  much 

92  See  on  the  other  hand  what  H.  v.  Mohl  says  in  objection  to  this  (Deber  den  angeblichen 
Gehalt  der  Stiirkekorner  an  Cellulose;  Bot.  Zeitg.  1859,  2-25,  ff). 

93  Sachs,  Concerning  the  substances  which  supply  the  material  for  the  growth  of  the 
cell  wall  (Pringsheim's  Jahrb.  Ill,  1863,  p.  183,  ff.}—  Behrens,  Die  Xectarien  derBlUten 
(Flora,  1879.  p.  244). 


364  THE  MICEOSCOPE  IN  BOTANY. 

protoplasmic  proteid  substance  and  in  this  small  imbedded 
grains  of  starch  (transitorily)  as  in  the  organs  of  secretion,  or, 
in  the  guard-cells  of  the  stomata,  the  blue  reaction  of  the 
starch  by  the  use  of  the  iodiue  reagent  will  be  concealed  by 
the  yellow  brown  of  the  nitrogenous  substances.  In  this  case 
in  order  to  make  the  starch  visible  lay  the  preparation  in  dilute 
potassium  hydroxide  which  will  dissolve  the  greater  part  of  the 
proteid  substance.  Wash  with  distilled  water,  neutralize  as 
much  as  is  necessary  with  weak  acid  and  then  add  the  potassium 
iodide  of  iodine  solution.  With  many  preparations  this  procedure 
is  not  necessary  since  sometimes  the  starch  colors  before  the 
plasma.  In  such  cases  a  dilute  iodiue  solution  may  be  suitably 
employed. 

B.    STAKCH  nr  CHLOBOPHYLII  GKAiNs.94 

The  green  color  of  the  chlorophyll  conceals  the  blue  re- 
action of  the  iodine  upon  the  starch  grains  contained  in  the 
chlorophyll,  and  the  chlorophyll  grains  receive  a  verdigris  green 
stain  from  the  iodine.  Very  small  granules  of  starch  thus 
embedded  appear  of  a  brown  color  after  treatment  with  iodine 
reagents  (v.  Mohl).  In  such  cases  in  order  to  make  the  starch 
grains  visible  it  is  necessary  to  lay  the  preparation  in  absolute 
alcohol  and  set  it  in  the  direct  sunlight  to  extract  the  green 
chlorophyll  coloring  matter.  Whenever  the  section  is  ready 
treat  with  potassium  hydroxide  to  swell  the  starch  grains,  wash 
out  the  alkali  with  water  and  add  solution  of  iodine  (Bohm). 
Or  treat  the  bleached  section  with  boiling  potassium  hydroxide, 
wash  out,  neutralize  with  weak  acid  and  add  dilute  iodine  solu- 
tion. Then  we  get  the  swollen  starch  grains  colored  a  distinct 
violet  blue.  Sometimes  the  preparation  becomes  still  more 
beautiful  when  it  has  lain  for  a  day  or  two  in  glycerine.  In 
filamentous  algee  (Conferva)  it  is  enough  often  to  add  the  iodine 
alone  to  make  the  large  starch  grains  visible  through  the  thin 
layer  of  chlorophyll  (v.  Mohl) . 

»*  H.  v.  Mohl,  Vermischte  Schriften,  p.  355.— H.  v.  Mohl  in  Bot.Zeit.,  1355,  pp.  110,  111.— 
Bohm  in  Sitzungsber.  d.  K.  K.  Acad.  Wien,  1857,  p.  21.— Sachs  in  Flora,  1862,  p.  166.— Sachs 
in  Pringsheim's  Jahrb.  Bd.  Ill,  1863,  pp.  186,  200. 


DEXTRINE.  365 


in.     DEXTRINE. 

Literature.  Sachs,  Ueber  einige  neue  mikrosk.-chem.  Re- 
actionsmethoden  (Sitzungsber.  d.  K.  Acad.  d.  Wiss.  Wien, 
Bd.  XXXVI,  1859,  p.  8,  /".)— -Sachs,  Mikrochem.  Uutersuch- 
ungen  (Flora,  1862,  p.  289,  /*.)  — Sachs,  Ueber  d.  Stoffe, 
welche  das  Material  zura  Aufbau  der  Zellhaute  liefern  (Prings- 
heim's  Jahrb.,  Bd.  Ill,  1863,  p.  183,/.)  — Dippel,  Mikrosk., 
Bd.  II,  p.  19. — Nageli  u.  Schwendener  Mikrosk.,  p.  475,  p. 
525.— W.  Nageli,  Die  Starkegruppe.— Poulsen,  Bot.  Mikro- 
kemi,  p.  54  (Trans,  p.  87). 

Dextrine  C6H10O5  or  C^H^O^  is,  in  the  dry  state,  an  amor- 
phous gum-like  mass,  in  solution  —  as  it  occurs  in  the  cells  — 
a  colorless  clear  fluid  which  is  produced  by  the  transformation 
of  starch.  This  transformation  seems  to  proceed  in  this  way: 
The  granular  starch  appears  first  to  be  changed  into  a  starch 
solution  (amylodextrine),  and  from  this  is  produced  the  charac- 
teristic dextrine.  It  has  not  yet  been  possible  to  trace  micro- 
scopically this  transformation.  A  wood  dextrine  produced  from 
cellulose  should  also  be  mentioned  (B^champ). 

The  single  method  known  at  the  present  time  for  microscopi- 
cally testing  dextrine  is  that  introduced  by  Sachs,  by  means  of 
Trommer's  reagent  (copper  sulphate  and  potash  solution,  see 
pp.  293  and  325) .  This  reagent  is  useful  for  testing  at  the  same 
time  the  other  soluble  carbohydrates  of  the  cell  contents  — 
grape  sugar,  cane  sugar,  etc.  —  it  often  likewise  affords  the 
means  of  studying  the  relative  quantity  of  these  elements  as 
well  as  their  distribution  in  the  interior  of  plants. 

Method  (Sachs)  ,95  In  order  to  prove  that  a  given  part  of  a  plant 
contains  dextrine  (or  sugar  or  both)  we  should  make  both  a  trans- 
verse and  a  longitudinal  section  which  is  at  least  two  or  three 
cell-layers  thick  and  lay  them  in  a  vessel  containing  a  concen- 
trated copper  sulphate  solution.  While  they  are  here  absorb- 
ing the  salt,  a  small  porcelain  dish  holding  about  5  or  6  cc. 

»  Sachs  in  Pringsheim's  Jahrb.,  Bd.  Ill,  p.  187,  /. 


366  THE  MICROSCOPE  IN  BOTANY. 

filled  with  strong  potash  lye  should  be  heated  to  boiling  over 
the  flame ;  then  with  the  forceps  take  the  section  out  of  the 
copper  sulphate  solution  and  dip  it  several  times  in  a  vessel  of 
water  and  lay  it  immediately  in  the  hot  alkali. 

If  the  cells  contain  grape  sugar  or  dextrine  there  will  be 
produced  almost  instantly  or  after  a  few  seconds  sometimes,  a 
beautiful  opaque  red  coloring  which  approaches  now  to  brick 
red  and  now  to  yellow.  This  coloring  comes  from  the  precipi- 
tate cuprous  oxide  produced  in  the  cells  which  under  high  mag- 
nification appears  in  the  form  of  small,  roundish  grains  in  the 
cells.  In  most  cases  the  reaction  is  so  brilliant  that  the  unaided 
eye  can  distinguish  the  presence  of  the  precipitate,  even  if  it 
occurs  only  in  single  cells.  Still  it  is  always  advisable  to 
accept  the  aid  of  the  magnification.  If,  on  the  contrary,  the 
section  contains  cane  sugar,  a  beautiful,  sky-blue  color  is  pro- 
duced on  dipping  the  section  in  potash  after  it  has  been  previ- 
ously saturated  with  the  copper  salt  solution  and  washed, 
which  color  belongs  to  a  clear  fluid  contained  in  the  cells.  There 
is  produced  no  precipitate  of  red  cuprous  oxide  by  boiling  in 
the  potash ;  the  fluid  remains  blue  and  diffuses  very  quickly 
through  the  potash. 

In  order  to  distinguish  if  the  reduction  of  the  red  cuprous 
oxide  comes  from  grape  sugar  or  dextrine,  a  section  of  the  same 
plant  in  which  the  reaction  has  been  produced  should  be  laid  in 
alcohol  of  90  or  95  per  cent.  Since  according  to  Pay  en  dex- 
trine is  itself  insoluble  in  alcohol  of  84  per  cent,  it  cannot  be 
extracted  from  the  cells  by  a  considerable  stronger  alcohol. 
The  section  then  containing  dextrine  should  show  the  red  re- 
action when  treated  as  described,  even  after  lying  for  several 
hours  in  the  alcohol.  If,  on  the  contrary,  the  cells  of  a  thin 
section  contain  grape  sugar  an  alcohol  of  90  to  95  per  cent  will 
dissolve  it,  and  after  ten  or  twenty-four  hours,  as  Payen  shows, 
that  alcohol  of  95  per  cent  will  dissolve  grape  sugar  itself.  If 
then  a  section  before  treatment  with  alcohol  reduces  with  cu- 
prous oxide,  and  after  that  shows  no  precipitate  of  red  grains, 
one  may  conclude,  according  to  Sachs,  that  the  reduction  of 
cuprous  oxide  is  due  to  grape  sugar  and  not  to  dextrine. 


VEGETABLE  MUCILAGE.  367 

With  the  inexperienced,  an  error  may  occur  both  in  the  re- 
action for  grape  sugar  and  dextrine  and  for  cane  sugar.  The 
outer  walls  of  wood  cells,  vessels,  etc.,  as  is  well  known,  are 
colored  orange-yellow  to  reddish  by  this  treatment,  parenchyma 
cells  and  very  young  wood  cells,  etc.,  blue  (see  p.  326). 
These  colors  to  the  unaided  eye  might  awaken  a  suspicion  that 
in  the  former  case  grape  sugar  or  dextrine  reaction  had  taken 
place,  and  in  the  latter  the  reaction  of  cane  sugar ;  .but  a  suffi- 
cient magnification  would  give  the  correct  information. 


IV.     VEGETABLE  MUCILAGE. 

Literature.  Vogel  und  Schleiden,  Ueber  d.  Amyloid,  eine 
neue  Pflunzeusubstunz  (PoggendorlTs  Annalen,  Bd.  XLVI, 
1839,  p.  327). — Kiitzing,  Grundz.  d.  philos.  Bot.,  Leipzig, 
1852,  Bd.  I,  p.  159,  /".—Cramer,  Botan.  Beitrage,  Zurich, 
1855,  p.  1,  ff. — Karsten,  Ueber  d.  Entstehung  des  Harzes, 
Wachses,  Gummis  und  Schleimes  durch  d.  assimil.  Thatigkeit 
d.  Zellmembranen  (Botan.  Zeitg.,  1857,  p.  313,  /".).—  Frank, 
Ueber  d.  anat.  Bedeut.  und  die  Entstehung  der  vegetab. 
Schleime  (Pringsheim's  Jahrb.,  Bd.  V,  I860,  pp.  161-200).— 
Hanstein,  Ueber  d.  Organe  der  Haiz-  und  Schleimabsonderung 
in  den  Laubknospen  (Bot.  Zeitg.,  1868,  p.  697,  ff.). — Barci- 
anu,  Ueber  d.  Blutentwicklung  der  Onagraceeu  (Schenk  und 
Lurssen's  Mitth.  aus  d.  Geb.  d.  Bot.,  Bd.  II,  p.  85). — Kirch- 
ner  und  Tollens,  Untersuchungen  iiber  d.  Pflanzenschleime 
(Ann.  Chem.  Pharm.,  Bd.  CLXXV,  p.  205).— Behreus,  Die 
]Srectarien  d.  Bliiten  (Flora,  1879,  p.  440,  ff.) — Szyszylowicz, 
Korallina  jako  odczynnik  mikrochemiczny  w  histyjologii  ros- 
linej  (Osobne  odbicie  z  Rozpran  Akad.  Uiniej  w  Krakowie, 
Bd.  X,  1882).— For  the  rest  see  the  treatises  cited  on  p.  327. 

This  widely  distributed  vegetable  substance  which  likewise 
bears  the  formula  C6HJOO5  or  C^H^O^  has  by  no  means  been  so 
thoroughly  studied  as  is  necessary  for  an  exact  knowledge  of 
it.  AVe  have  already  explained  (p.  328,  ff.)  that  it  is  fre- 
quently produced  by  a  metamorphosis  of  the  cell  membranes ; 
that  there  are  even  various  transformations  of  essential  cellu- 


368  THE  MICROSCOPE  IN  BOTANY. 

lose  into  mucilage  which  are  accounted  for  with  the  greatest  diffi- 
culty. Other  mucilage  may  doubtless  be  indebted  to  starch  for 
its  origin  (mucilage  of  the  orchid  bulbs)  ;  still  others  present  a 
mixture  of  mucilage  and  gum.  Generally,  it  is  a  matter  of  no 
small  difficulty  to  keep  the  gums  and  mucilaginous  substances 
separate.  In  a  general  way  it  may  be  said  that  all  mucilage 
gives  a  blue-violet  and  sometimes  a  yellow  color  with  iodine, 
and  with  oxalic-nitric  acid ;  that  the  gums  give  no  color  with 
iodine  but  are  transformed  into  mucic  acid  by  nitric  acid. 

In  the  present  condition  of  theories  concerning  the  nature  of 
vegetable  mucilage  it  would  be  hazardous  to  undertake  a 
classification  of  them ;  the  following  on  that  account  may  be 
looked  upon  as  somewhat  imperfect.  Of  the  mucilages  we  dis- 
tinguish : 

1.  Characteristic  Mucilage.     In  water  it  is  either  insoluble, 
soluble  or  simply  swells.     Alcohol  precipitates  it  from  an  aque- 
ous solution.     It  colors  blue  or  violet  with  iodine,  or  iodine  and 
sulphuric  acid ;  with  Hanstein's  aniline  mixture  red  or  reddish 
(if  at  all   colored),  and  yields  oxalic  acid  when  treated  with 
nitric  acid. 

(a)  Mucilage  from  Cellulose  (e.  g.,  roots  of  Symphytum). 
(6)  Mucilage  from  Starch  (e.  g.,  Orchis  bulbs). 

2.  Amyloid.    Mostly  soluble  in  water ;  with  iodine  it  gives  a 
yellow,  yellow  red  and  also  a  blue  color.     From  an  aqueous  so- 
lution alcohol  precipitates   a  jelly-like  substance  which  takes 
a  blue  color  from  iodine.     With  Hanstein's  aniline  it  gives  a 
beautiful  red  or  scarlet-red  color.     Perhaps  nearly  related  to  I  b. 

3.  Lichenin  (lichen  starch).    Swells  but  does  not  dissolve  in 
water ;  precipitates  by  alcohol ;  iodine  alone  colors  it  yellow, 
green  or  blue. 

4.  Gum  Mucilage.     Swells  or  dissolves  in  water,  giving  with 
iodine  no  color  or  yellowish  to  reddish;  indifferent  to  most 
anilines  ;  treated  with  nitric  acid  it  yields  mucic  acid,  or  oxalic 
with  mucic  acid.96 

98  Giraud  (according  to  Husemann,  Pflanzenstoffe,  Bd.  I,  p.130)  divides  plant  mucilage 
into  two  kinds,  those  producing  pectin  (gum  tragacanth)  and  those  not.  The  latter  are 
separated  into  those  which  are  changed  into  an  insoluble  form  by  acids  and  those  not  pre- 
cipitated by  acids. 


VEGETABLE  MUCILAGE.  369 


1 .     Characteristic  Mucilage. 

This  is  very  widely  distributed  through  the  vegetable  king- 
dom, being  formed,  for  example,  in  the  swelling  secondary 
layers  in  the  parenchyma  of  the  cotyledons  of  many  plants, 
and  in  many  muciparous  roots,  as  of  /Symphytum,  Orchis,  etc. 
Whether  the  amyloid  which  was  first  detected  by  Schleidcn 
and  Vogel  in  the  cotyledon  of  Lupinus,  Tropceolum,  etc.,  is 
to  be  ascribed  to  it,  or  represents  a  type  of  mucilage  of  its 
own,  must  remain  temporarily  doubtful.  I  have  recently  ex- 
pressly demonstrated  that  in  the  neighborhood  of  muciparous 
cells,  often  in  them,  crystals  or  bundles  of  crystal-needles  of 
calcium  oxalate  are  found.97  I  then  expressed  the  opinion 
that  they  might  stand  in  some  relation  to  the  mucilage  exist- 
ing there  though  one  might  not  be  able  to  say  how.  The 
probability  of  this  interdependence  is  considerably  augmented 
by  the  fact  that  plant  mucilage  is  chemically  transformed  into 
oxalic  acid  by  tin  oxydizing  medium,  as,  for  example,  nitric 
acid.  Frank98  observed  some  time  ago  that  in  the  formation 
of  the  orchis  mucilage  the  oxalic  needles  found  in  that  vicin- 
ity slowly  diminished. 

Many  mucilages  belonging  to  this  group  are  colored,  with 
iodine  alone  or  with  iodine  and  sulphuric  acid,  a  blue  or  violet, 
giving  the  reaction  of  cellulose."  With  others  this  is  not 
the  case,  as  this  treatment  produces  a  yellow  or  yellowish 
color.100  Hanstein's  aniline  mixture  gives  to  most  of  the 
mucilages  a  full,  red  color  which  sometimes  has  a  tinge  of 
purple.  Successive  treatment  with  creosote,  zinc  chloride 
and  aniline  will  cause  a  reddening  of  the  mucilage  (Barciauu). 
(The  effect  of  aniline  on  mucilage  has  been  but  little 
studied.) 

Quite   recently  Szyszylowicz  has   designated   coralline  as  a 


»?  Behrens,  Die  Xectarien  der  Bliiten  (Flora,  1879,  p.  450). 

88  Frank  in  Pringsheim's  Jahrb.,  Bd.  V,  p.  181. 

89  Kiitzing,  Grundz.  d.  philos.  Bot.,  Bd.  1,  p.  195.— Frank,  I.  c.,  pp.  168,  181. 
"o  Frank,  I.  c.,  pp  1653, 165, 167.— Hanstem  in  Bot.  Zeit.,  1868,  p.  700,  etc. 

24 


370  THE  MICROSCOPE  IN  BOTANY. 

preferable  reagent  for  vegetable  mucilage,  and  indeed  it  is 
possible  by  means  of  this  reagent  to  distinguish  the  mucilage 
which  comes  from  starch  from  that  which  is  produced  from  cel- 
lulose. 

Co'Mlline  or  rosalic  acid, a  red  powder  with  greenish,  metallic 
luster,  dissolves  in  very  dilute  alcohol  and  in  alkali  salts,  but  not 
in  water,  with  a  beautiful  coral-red  color.101  Szyssvlowicz  pre- 
pared it  from  carbolic  acid  by  the  action  of  sulphuric  acid  in 
the  presence  of  oxalic  acid.  It  then  contained  some  aniline 
mixed  with  it.  He  dissolved  it  in  sodium  carbonate  (probably 
because  an  alcoholic  solution  would  produce  the  precipitation 
of  the  muciluge).  The  solution  is  purple-red  and  unchange- 
able in  the  light. 

According  to  Szyszylowicz  this  reagent  gives  to  starch 
mucilage  a  permanent  red,  the  color  not  being  destroyed 
even  by  prolonged  boiling  in  alcohol ;  protoplasm  and  the 
cell  walls,  however,  remain  perfectly  colorless.  Cellulose 
mucilage  is  likewise  colored,  but  the  color  may  be  removed  by 
cold  and  especially  by  hot  alcohol.  Many  colored  preparations 
of  starch  mucilage  may  be  preserved,  preferably  in  Canada  bal- 
sam ;  others  on  the  contrary  cannot. 

2.     Amyloid. 

This  is  also,  as  has  been  stated,  very  like  typical  mucilage. 
It  is  often  very  sensitive  to  the  action  of  iodine  reagents. 
With  Hanstein's  aniline  mixture  it  yields  a  red  or  scarlet- 
red  color.102  Hanstein  appears  to  suppose  that  there  are  sev- 
eral kinds  of  amyloids,  on  what  grounds  it  is  not  very  clear 
to  me.  Whether  what  Hanstein  calls  "collagene"  is  to  be  in- 
cluded in  this  or  rather  among  the  gum  mucilages  is  also 
doubtful.  He  once  declared  collagene103  to  be  of  those  cellu- 
lose-like bodies  which  are  finally  transformed  into  mucilage, 
simply  b}r  absorbing  water,  not  observing  if  it  chemically  stood 

101  This  substance  is  to  be  had  everywhere,  since  in  recent  times  it  lias  come  into  general 
use  in  the  chemical  laboratory  as  an  important  test  in  volumetric  determinations  in  the 
place  of  litmus. 

102  Hanstein,  I.e.,  a.  v.  O. 

103  Hanstein,  1.  c.,  p.  700,  note. 


GUM.     (ARABIN,  BASSORIN.)  371 

nearer  to  this  or  another  amyloid ;  afterwards  he  mentioned10* 
that  gum  mucilage  was  produced  essentially  out  of  wall-build- 
ing amyloid  substances.  Finally,  he  was  never  able  to  bring 
the  colliigene  layer  to  the  red  reaction  with  aniline,  as  it  is  al- 
ways possible  to  do  with  amyloid. 

3.     Lichnin  or  Lichen  Starch. 

This  appears  to  stand  very  near  to  starch.  It  occurs  ii\ 
many  lichens  ( Cetraria,  Cladonia  Ramalina)  and  in  marine 
algae  (Delesseria)  in  the  form  of  small  grains  or  as  a  jelly-like 
mass.  Lichnin  swells  but  does  not  dissolve  in  water.  Alco- 
hol precipitates  it  unchanged,  but  caustic  alkalies  dissolve  it. 
With  iodine  alone  it  colors  yellow,  grey  or  blue.  It  is  dis- 
solved by  chlor-iodide  of  zinc  and  cuprammonia. 

4.     Gum  Mucilage* 

We  designate  by  this  expression  several  slimy  substances 
which  in  part  may  be  a  mixture  of  gum  and  mucilage,  and 
in  part  simple  substances  standing  between  the  gums  and 
mucilages.  Although  they  often  occur  they  are  but  little 
known.  To  this  group  belong,  for  example,  the  mucilage 
of  many  fuci,  the  mucilage  of  the  quince,  the  mucilage  of 
the  seeds  of  flax,  of  Plantago  psyUium  and  P.  lanceolate 
and  the  althea  mucilage.  Here  also  perh-ips  the  colliigene  of 
Haustein  may  be  best  placed.  With  nitric  acid  it  is  de- 
composed into  mucic  and  oxalic  acid,  white  the  true  gums  give 
only  mucic  acid.  According  to  Szyszylowicz  the  gums  do  not 
thoroughly  stain  with  coralline;  they  do  color  with  it  more  or 
less,  the  intensity  and  permanency  of  the  color  depending  upon 
the  relations  of  the  two  substances. 


V.     GUM.     (ARABIN,  BASSORIX.) 

Literaiu  e.  Mcyen,  Pflanzenpathologie,  1841,  pp.  229-255, 
— Mohl,  Outers,  iiber  d.  Entstehungsweise  des  Traganthgummi 
(Bot.  Zeitg.,  1857,  p.  33,  ff.) — Karsten,  Oeber  d,  Entstehung 

l«*  Hanstein,  1.  c.,  p.  774. 


372  THE  MICROSCOPE  IN  BOTANY. 

d.  Harzes,  Wachses,  Gummis,  etc.  (icZ.  1857,  p.  313,  /*.).— 
Hofmeister,  Ueber  d.  zu  Gallerte  aufquellenden  Zellen,  etc. 
(Ber.  d.  sachs.  Gesellsch.  d.  Wiss.  zu  Leipzig,  Bd.  X,  1858, 
pp.  18-36). —  Trecul,  Production  de  lagomme  chez  le  Cerisier, 
le  Prunier,  1'Amandier,  1'Abricotier  et  la  Pecher  (Proces-ver- 
baux  des  seances  de  la  soc.  philomat.  pend.  1'annee,  1862,  voir 
aussi  Journal  de  I'lnstitut,  1862,  p.  241). —  Wigancl,  Ueber 
d.  Desorganisation  d.  Pflanzcnzclle,  insbes.  liber  d.  physiol. 
Bedeutung  von  Gummi  und  Harz  (Pringsheim's  Jahrb.,  Bd. 
Ill,  1863,  pp.  115-186). — Kraus,  Ueber  den  Ban  d.  Cycadeen- 
tiedern  (id.  Bd.  IV,  1865,  pp.  328-329).— Frank,  Ueber  d. 
anat.  Bedeut.  n.  d.  Entstehnng  der  veget.  Scbleitne  (id.  Bd. 
V,  1866,  pp.  161-200).— Miiller,  Unters.  iiber  die  Vertheilung 
d.  Harze,  ather.  Oele,  Gnrnmi  und  Gummiharze,  etc.  (id. 
Bd.  Y,  1866,  p.  397,  ff.).— Hofmeister,  Lchre  v.  d.  Pflzelle., 
Leipzig,  1867,  p.  234,  ff. — Hanstein,  Ueber  d.  Organe  der 
Harz-  und  Scbleimabsondernng  in  den  Laubknospen.  (Botan. 
Zeitg.,  1868,  p.  697,  ff.). — Sorauer  in  Landwirthschaftl. 
Versuchsst.,  1872,  p.  454. — Prillieux,  Etude  sur  la  format,  de 
la  gomme  dans  les  arbres  fruitiers  (Ann.  des  sc.  nat.  Bota 
nique,  6e  ser.  t.  I,  1875,  pp.  176-200).— Szyszylowicz,  Koral 
lina  jako  odczynnik  rnikrocbemiczny  w  histyjologii  rosliunej 
(Osobne  odbicie  z.  Rozpran  Akad.  Umiej  \v  Krakowie  t.  X).105 
The  numerous  extremely  heterogeneous  substances  which  are 
united  under  this  name,  and  to  which  may  be  ascribed  the  formula 
C6H10O5  or  multiples  of  it,  occur  in  plants  as  mucilaginous,  col- 
orless, yellow  or  brown  masses  which  are  either  soluble  in  water 
and  are  then  called  Arabin  or  Cerasin  (gum  arable,  cherry  gum), 
or  are  insoluble  in  water  and  are  then  named  Bassorin  or  Adra- 
gantin  (gum  tragacanth).  The  previously  named  pectinaceous 
substances  probably  also  belong  with  these  as,  according  to  the 
opinion  of  many  chemists,  pectose  is  nothing  else  than  changed 
arabin.  On  the  other  hand  it  appears  that  many  species  of  bas- 
sorin  and  adragantin,  for  example  gum  tragacanth,  are  strongly 
impregnated  with  pectose.  The  gums,  like  the  mucilages,  have 
been  but  little  studied  heretofore,  so  that  the  proposal  of  the 

106  Compare  also  the  literature  cited  on  p.  327  and  p.  343, /.,  the  latter  in  reference  to 
pectin  metamorphosis. 


GUM.     (ARABIN  BASSOKIN.)  373 

chemists  to  unite  together  temporarily  in  one  category  all  pec- 
tinaceous  substances,  mucilages  and  gums,  and  to  designate 
them  r  jelly-forming  carbohydrates,"  has  much  in  its  favor. 

Preliminarily  it  may  be  said  only  that  the  gums  in  a  restricted 
sense  are  distinguished  from  the  mucilages  in  that  they  give  no 
reaction  with  iodine  reagents  and  that  when  treated  with  nitric 
acid  they  do  not  change  as  those  do  into  oxalic  acid  but  into  mucic 
acid.  Recently  it  has  been  stated  by  Szyszylowicz  that  in  op- 
position to  true  mucilage  they  do  not  color  with  coralline. 

They  occur  either  alone  or  mixed  with  mucilage  (mucilage 
gum)  or  they  form  a  more  or  less  intimate  mixture  with  the 
resins  (resin  gums) .  Of  the  occurrence  of  the  latter  kind,  some 
very  beautiful  examples  have  been  described  by  Hanstein106  in 
the  young  leaf  buds. 

The  gum  masses  are  either  pure  secretions  such  as  are  poured 
forth  in  a  pathological  condition  of  the  plant,  as,  for  example, 
the  cherry  gum,  or  that  peculiar  formation  of  gum  in  the  wood 
of  the  orange  tree  which  in  Italy  is  known  as  the  "  Mai  della 
gomma."107  But  in  other  cases  the  formation  of  the  gum  is  a 
normal  process.  Then  it  will  commonly  be  poured  out  into  re- 
ceptacles, gum  conduits. 

The  production  of  gum  trngacanth  out  of  parts  of  the  cell  wall 
was  first  studied  by  v.  Mohl  and  rightly  explained  (see  p.  329). 
Karsten  regarded  all  kinds  of  gums  as  the  product  of  the  cell 
wall.  Trecul  and  Frank  agree  with  this  but  Wigand  made  va- 
rious objections.  Trecul  allows  that  sometimes  the  contents 
of  the  vessel  in  part  participate  in  the  formation  of  the  gums 
and  describes  reservoirs  for  the  formed  gum.  Frank  held  that 
the  pouring  out  of  gum  was  always  a  symptom  of  disease,  the 
cause  of  which  lay  in  the  heaping  up  of  plastic  matter  in  a  cer- 
tain place  whereby  the  equilibrium  of  the  interior  of  the  plant 
is  disturbed. 

Prillieux  agrees  with  Trecul  and  finds  that  gum  is  formed 
both  in  gum  conduits  and  in  the  vessels  the  latter  having  in  the 

"8  Hanstein  in  Bot.  Zeitg.,  18G8,  p.  704,  ff. 

107  Novellis,  11  male  della  gomma  degli  agrnmi(L'AgricolturaMeridionale,Portici  1879; 
Cf.  des  Turf.  Besprechuug  in  Bot.  Ceiitralbl.  Bd.  II,  1880,  p.  469 /.; 


374  THE  MICROSCOPE  IN   BOTANY. 

cherry  tree  both  pitted  and  spiral  thickenings.  It  is  sometimes 
produced  from  starch.  According  to  Hofmeister108  it  is  an  im- 
proper expression  to  speak  of  the  formation  of  gum  by  the 
disorganization  of  the  cell  wall,  since  it  existed  before  the  de- 
struction of  the  walls  as  cell  contents  and,  together  with  the 
transformed  walls,  forms  the  secretion.  Gum  substances  appear 
never  to  occur  in  the  cell  sap. 

A.  Arabin  and  Oerasin.      Cherry  gum  is  colored  yellow 
with  iodine  and    chlor-iodide  of  zinc,  brownish  with    potash, 
yellow,  or  in  earlier  stages,  a  lively  violet  with  muriatic  acid 
(Prillieux)  and  dissolves  in  water.109 

The  gum  in  the  conduits  of  the  pinnae  of  the  Cycadece  is  not 
colored  by  chlor-iodide  of  zinc  and  forms  flocculent  masses  with 
alcohol.110  Coralline  leaves  the  gum  uncolorccl.  All  walls  at- 
tacked by  gummosis  frequently  become  violet  with  aniline,  ac- 
cording to  Hanstein. 

B.  Bassorin.      Admgantin.      According   to   Mohl,    gum 
tragacanth  is    not    colored  by  iodine  or  chlor-iodide  of  zinc. 
According  to  Prillieux111  there  may  be  noticed  in  it  layers  of 
cell  walls  which    color  blue  violet  with  chlor-iodide  of  zinc. 
According  to  this  author  it  consists  of  a  mixture  of  cellulose  and 
of  a  mucilaginous  substance  which  is  interposed  between  the 
very  thin  layers  of  the  cellulose. 

C.  The  Mucilage  of  Flax  seed  is  a  gummy  mucilage  which 
behaves  like  bassorin,  swelling,  but  not  dissolving,  in  water  and 
not  coloring  blue  with  iodine  and  sulphuric  acid,  at  most  only 
yellow.      It   is    insoluble   in    cuprammonia   not   even     swell- 
ing in  it.     The  mucilage  of  Plantago  psyllium  is  similar  but 
dissolves  with    ease    in    cuprammonia,    but    does    not    color 
blue  with  iodine  and  sulphuric  acid.     The  gummy  mucilage  of 
the   root   of    Althaea   officinal  is   behaves    in    the   same  way. 

108  Hofmeister,  Pflanzenzelle,  p.  234. 

189  According  to  Prillieux  (Ann.  Sc.  Nat.  1.  c.  p.  182  /.)  there  is  found  in  the  vessels  of  the 
cherry  wood  a  kind  of  gum  (Cerason)  different  from  Cerasin  which  is  not  soluble  in  water 
and  is  colored  rose  red  with  iodine  and  snlphuric  acid.  It  is  very  much  like  the  so-called 
Eugelacin  of  Kiitzing  found  in  many  algae.  It  is  colored  violet  with  muriatic  acid. 

«°  Kraus  in  Pringsheim's  Jahrb.,  Bd.  IV,  p.  329. 

»»  Prillieux,  1.  c.  p.  181  /. 

112  Frank,  I.  c.,  p.  161-167. 


IXULIN.  375 


VI.     INULIX. 

Literature.  Rose,  Ueber  eine  eigenth.  vegetab.  substanz 
(Gehlen's  neues  Jouru.  d.  Cliem.,  Bd.  Ill,  1804,  pp.  217-219) 
— Payen,  Extrait  d'un  mem.  lu  a  1'Institut  sur  line  nouv.  subst. 
trouvee  dans  les  tuberc.  des  dahlias  (Journ.  de  Pharm.,  t.  IX, 

1823,  pp.  383-392). — Payen,  Observ.  sur  Panalyse  des  tuberc. 
de  I'Helianthus  tuberosus  (Ann.  de  Chun,  et  de  Phys.,  t.  XXVI, 

1824,  pp.  98-106).— Meyen,  Neues  Syst.  d.  Pfl.-Phys.,  Bd.  II, 
1838,  pp.  281-285. — Payen,  complement  d'un  memoire,  etc. 
(Ann.  des  sc.  nat.  2e  ser.,  t.  XIV,  Botanique,  1840,  p.  85,^*.). 
— Cramer,  Ueber  d.  Verhalten  des  Kupferoxydammoniaks  zur 
Pflanzenzellmembran,  zu  Starke,  Inulin,  etc.  (Vierteljahrsschr. 
d.  nat.  Ges.  Zurich,  Bd.  Ill,  1858,  pp.  1-22).— Mohl,  Unters. 
des  Pflanzengew.  mit  Hilfe  des  polar.  Lichtes  (Botan.  Zeitg., 
1858,  p.  \,jf.). — Hartig,  Entwieklungsgesch.  d.  Pflkeims,  Lpz., 
1858,  p.  117. — Schacht,  Ueber  d.  Inulin  (Sitzungsber.  niederrh. 
Gesellsch.  zu  Bonn,  1863,  pp.   174-177). — Sachs    (id.,    pp. 
177-180).— Sachs,  Ueber  d.  Spharokrystalle  des  Inulins  (id., 
1864,  pp.  9-11'). — Sachs,  Ueber  d.  Spharokrystalle  des  Inulins 
und  dessen  Mikrosk.  Xachweisung  in  den  ZeHen  (Botan.  Zeitg. 
1864,  p.  25,^*.). — Prantl,  Das  Inulin.  Ein  Beitrag  zur  Pflanzen- 
physiologie,  Munchen,  1870. — Dragendorf,  Materialien  zu  einer 
Monographic  des  Inulins,  Petersburg,  1870. — Kraus,  Beobacht. 
tiber  d.  Vorkommen  des  Inulins  (Sitzungsber.  naturf.  Gesellsch. 
zu  Halle  27,  F«b.,  1875. )113 

Inulin,  called  also  Alantin,  Helenin,  Dahlin,  has  the  formula 
C0H10O5  or,  according  to  Kiliani,  C^H^O^.  It  occurs  in  the 
underground  organs  of  the  Composites  (  Taraxacum  officinale, 
Hdianthus  tuberosus,  Georgina  variabilis,  Inula  Helemum, 
Eupatorium  cannabium,  etc.)  in  Campanula  rapunculoides,  in 
some  other  phanerogams  and  in  numerous  cryptogams  (Aceta- 
bularia  mediterranea,  Elaphomyces  granulatus,  etc) .  In  the  first 
named  plants  it  always  occurs  in  the  parenchyma  cells  (Sachs, 

113  Prantl  gives  a  full  list  of  the  literature,  also  of  the  chemical,  I.e.  pp.  67-72;  of  the  chem- 
ical after  1S70,  see  Huseinann,  Pflanzenstofle,  Bd.  1, 18S2,  p.  137. 


376  THE  MICROSCOPE  IN  BOTANY. 

Berg,  Prjintl),114  and  indeed  in  the  cell  sap,115  in  the  form  of  a 
concentrated  solution  which  is  pretty  strongly  refractive.  It  is 
almost  insoluble  in  cold  water,  but  dissolves  in  water  warmed 
to  from  50°  to  55°. 116  Inulin  is  insoluble  in  alcohol,  ether, 
glycerine,  fatty  or  essential  oils,  the  first  precipitating  it  from 
an  aqueous  solution.  On  this  is  founded  the  one  only  method 
of  microscopically  testing  it.  Strong  acids  dissolve  inulin 
after  it  first  becomes  transparent,  likewise  potash  lye  and  zinc 
chloride  solution.  Cuprammonia  also  dissolves  inulin,  the 
solution  beginning,  according  to  Cramer,  not  on  the  surface 
but  in  the  center  of  the  spherical  crystal.  If  the  inulin  be 
slowly  crystallized  from  a  solution,  it  forms  the  spherical  crys- 
tals of  a  most  highly  characteristic  structure,  which  grow  by  a 
process  of  superposition.117  Iodine  reagents  do  not  color  inu- 
lin, as  would  be  self-evident,  since  it  is  incapable  of  swelling.118 
If  a  microscopic  section  containing  precipitated  inulin  be  boiled 
for  some  minutes  in  water  having  a  trace  of  muriatic  acid, 
a  considerable  quantity  of  cuprous  oxide  may  be  reduced  inside 
the  cells  by  a  copper  sulphate  and  potash  process  described  on 
pp.  293  and  305.  This  cannot  be  done  with  a  fresh  section, 
only  with  one  which  has  previously  been  boiled  in  acidulated 
water.  By  this  process  the  inulin  is  transformed  into  glycose 
(levulose).119  Heated  on  the  platinum  slip,  inulin  develops  a 
vapor  which  strongly  smells  like  burning  sugar.120 

Method  of  microscopical  examination  (Sachs).121  From  tis- 
sue containing  inulin  should  be  cut  sections  more  than  one  cell 
layer  thick  and  then  covered  with  a  large  drop  of  alcohol.  This 
produces  a  milky  precipitate.  After  some  minutes,  however, 
the  preparation  clarifies  itself  by  the  formation  of  spherical 
crystals.  Now  by  dipping  it  in  water  the  small  granules  disap- 
pear and  the  spherical  crystals  alone  remain  behind,  the  irlam- 

11*  Prantl,  1.  c.,  p.  39. 

115  Mohl,  Bot.  Zeitg.,  1858,  p.  17.— Schacht,  Sitzungsber.  Bohn,  1S63,  p.  175.— Sachs,  id., 
p. 177. 

"6  Sachs,  Botan.  Zeitg.,  1864,  p.  78. 

"TXageliin  Sitzungsber.  K.  Bayer,  Acad.  d.  Wiss.,  Munchen,  1862,  Bd.II,  p.  3U,ff.— 
Sachs,  L  c.,  p.  80. 

us  Prantl,  1.  c.,  p.  30. 

i">  Sachs,  Sitzungsber.,  Bonn,  1863,  p.  178, 1864,  p.  10. 

12°  Sachs,  I  c.,  p.  78. 

121  Sachs,  I.  c.,  p.  85. 


GRAPE  SUGAR,  GLYCOSE. 


377 


inated  structure  becoming  more  distinct  (Fig.  133). 
crystals  become  much  larger,  often  breaking 
through  the  tissue,  if  we  put  large  organs  con- 
taining inuliu  for  a  long  time,  days  and  weeks, 
in  alcohol  and  glycerine  and  then  prepare  the 
sections  from  them.  Also  by  allowing  such  organs 
to  dry,  one  can  detect  inulin  in  them  in  the  form  of 
spherical  crystals,  after  the  preparation  of  the 
sections  .  According  to  Sachs  the  external  appear- 
ance of  the  inulin  crystals  is  sufficient  to  identify 
them  as  such,  but,  according  to  Prantl,  a  more 
exact  testing  of  their  qualities  as  given  above  is 
indispensable. 


The 


FIG.  133. 


VII.     GEAPE  SUGAR,  GLYCOSE. 

Literature.  Sachs,  Ueber  eiuige  neue  mikrosk.-chem.  Re- 
actionsmethoden  (Sitzungsber.  d.  K.  Acad.  d.  Wiss.  Wien, 
Bd.  XXXYI,  1859,  p.  5,  ff.).—  Sachs,  Mikrochem.  Unters. 
(Flora,  1862,  p.  -289,  ff.).— Sachs,  Ueber  d.  Stoife,  welche  d. 
Material  z.  Aufbau  d.  Zellwand  liefern  (Pringslieirn's  Jahrb., 
Bd.  Ill,  1863,  p.  183,  jf.)- 

Grape  sugar,  also  called  glycose  or  starch  sugar,  C6  H^  O6, 
very  frequently  occurs  in  plant  tissues  and  almost  without  ex- 
ception in  aqueous  solution,  and  mostly  in  connection  with 
solutions  of  cane,  or  other  kinds  of  sugar.  It  arises  as  a  trans- 
formation product  of  other  carbo-hydrates  chiefly  indeed  of 
starch  (starch  sugar)  and  in  this  form  appears  to  travel  through 
the  interior  of  plants. 

The  single  method  for  the  testing  of  grape  sugar,  as  is  that  of 
cane  sugar  and  dextrine,  is  based  on  the  reaction  of  copper 
sulphate  and  potash  discovered  by  Trommer  and  modified  by 
Fehling.  It  was  introduced  into  microscopy  by  Sachs. 

Impregnate  .the  tissues  containing  the  glycose  with  copper 
sulphate  solution  and  add  thereto  cold  potash  lye  in  excess  so 
as  to  produce  a  beautiful  blue  fluid.  Directly,  if  cold,  sooner 


Nach  Sachs,  I.  c.,  Taf.  II,  fig.  5. 


378  THE  MICROSCOPE  IN  BOTANY. 

by  boiling,  there  appears  a  copious  display  of  reduced  copper 
oxide  which  is  colored  a  beautiful  brick  red.  Under  the  mi- 
croscope the  precipitate  appears  like  that  of  dextrine ;  the 
grains,  however,  are  larger  and  collected  in  many  large  flocculent 
masses  which  is  not  the  case  with  dextrine.  The  manipulation 
to  be  followed  in  this  reaction  is  the  same  as  that  described  on 
page  365,  for  dextrine;  for  this  reason  it  should  be  remarked 
here,  that,  as  in  the  case  of  dextrine,  the  section  to  be  tested 
for  grape  sugar  should  have  a  thickness  greater  than  that  of  a 
single  layer  of  cells,  else  will  the  fluid  cell  contents  escape  and 
the  desired  reaction  will  not  take  place. 

In  place  of  a  pure  copper  sulphate  solution  one  containing  a 
tartaric  acid  salt  may  be  employed  (Fehling's  solution).  The 
latter  is  prepared  by  dissolving  one  part  by  weight  of  copper 
sulphate,  and  five  parts  of  potassium  sodium  tartrate  in  eight 
parts  of  water  afterwards  keeping  the  solution  in  the  dark. 

VIII.     CANE  SUGAR,  SACCHAROSE. 

Literature.     The  same  as  in  the  grape  sugar. 

As  is  the  grape  sugar  so  is  the  cane  sugar  or  saccharose, 
C12  H^  Ou,  a  widely  distributed  plant  substance  which  presents 
itself  in  the  cells  likewise  as  a  clear  solution. 

The  method  for  microscopical  testing  as  given  by  Sachs  is  the 
same  as  that  for  grape  sugar.  Treat  a  section  containing  cane 
sugar  with  Tronimer's  or  Fehling's  solution  and  then  add  potash 
lye  and  there  will  appear  a  beautiful  blue  coloring,  but  no  re- 
duction of  copper  oxide  takes  place  even  after  a  short  boiling. 
The  appearance  of  the  blue  coloring  in  the  cold  liquid  is  quite 
characteristic  and  is  indeed  definitely  perceptible  even  with 
quite  thin  sections. 

The  reduction  of  copper  oxide  shows  always  the  presence  of 
grape  sugar  or  of  dextrine,  but  with  cane  sugar  a  reduction 
never  occurs. 

Concerning  testing  for  a  mixture  of  these  substances  with  each 
other  or  with  albuminous  matter  see  Sachs  in  the  proceedings 
of  the  Imperial  Academy  of  Sciences,  Vienna,  Vol.  XXXVI, 
p.  10,  f. 


ALBUMINOUS  MATTER.     PROTEIDS.  379 


IX.     ALBUMINOUS  MATTER.     PROTEIDS. 

Among  nitrogenous  substances  in  plants,  albuminous  or  pro- 
teid  matter  holds  the  chief  place.  It  is  lacking  in  no  living 
cell  and  all  vital  processes  are  intimately  connected  with  it. 
Chemically  it  is  characterized  by  its  components  of  carbon, 
oxygen,  nitrogen  and  hydrogen,  with  a  less  but  constant  quan- 
tity of  sulphur.  As  to  the  rest  a  satisfactory  chemical  formula 
has  not  yet  been  made.  Perhaps  this  is  in  general  im- 
possible, since  the  albuminous  substances  met  with  in  plants 
are  probably  not  individuals  in  the  chemical  sense.  Such  a 
formula  has  been  long  desired  for  protoplasm  for^instance. 
The  recently  published  chemical  analyses  of  the  protoplasm 
of  ^Ethalium  septicum123  by  Rodewald  show  it  to  consist  of 
a  large  number  of  different  organic  and  inorganic  chemical 
elements. 

Albuminous  substances  occur  in  cell  contents ;  they  are  solid 
or  plastic-viscous,  often  indistinguishable  from  a  fluid.  They 
are  insoluble  in  absolute  alcohol,  soluble  or  insoluble  in  water. 
They  are  mostly  colorless,  rarely  yellowish,  and  still  more 
'rarely  reddish  or  bluish. 

As  universal  qualities  of  albuminous  substances,  which  make 
their  recognition  easily  possible  under  the  microscope,  are  the 
following :  that  they  take  a  yellow  or  dark  yellow,  or  brown 
color  from  any  weak  solution  of  iodine,  the  color  being  more 
intense  than  that  of  the  surrounding  iodine  solution  ;  thai  they 
enter  into  a  dark-yellow  combination  with  nitric  acid  which  was 
invested  by  Mulder  with  the  name  of  xanthroproteid  acid ;  that 
they  take  a  beautiful  violet  color  with  copper  sulphate  and 
potassium ;  that  they  give  a  beautiful  rose-red  with  Millon's 
reagent ;  that  they  become  red  with  a  solution  of  sugar  and  the 
aid  of  sulphuric  acid  ;  that  they  are  unchanged  by  Hanstein's 
aniline  solution.  Dead  but  not  living  albuminous  matter  gener- 
ally may  be  stained  with  coloring  substances,  as  carmine  solu- 
tions, haematoxylin,  etc. 

"3  Reinke,  Ueber  d.  Zusaramensetz.  d.  Pro  topi.  v.  JEthal.  sept.  Gottingen  1880.— Reinke 
n.  Rodewald,  Studien  iiber  d.  Protoplasraa,  I,  Die  Chem.  Zusamniensetz.  des  Protopl.  v. 
sept.  (Unters.  aus  d.  Bot.  Laborat.  d.  Uuiv.  Gottingen,  Heft  II,  1881,  pp.  1-75). 


380  THE  MICROSCOPE  IN  BOTANY. 

A  chemical  classification  of  proteid  substances  has  indeed 
frequently  been  attempted,  but  heretofore  with  no  very  satis- 
factory results.  Ritthausen,124  one  of  the  best  of  those  who 
know  these  substances,  divides  them  into  albumin  (plant  albu- 
min), plant  casein  (legumin,  conglutin  and  glutencasein)  and 
paste-like  proteids  (gliadin,  mucedin  and  glutenfibrin).  Pfeff- 
er125  provisionally  holds  every  chemical  classification  to  be  in- 
sufficient and  with  A.  Mayer126  classifies  albuminous  bodies  as 
reserve  and  functional  proteid  substances.  This  classification  is 
the  foundation  of  the  following  representation. 

I.    RESERVE  PROTEID  SUBSTANCES. 
(Proteid  grains,  Aleuron,  Gluten  Meal.) 

Literature.  Hartig, Ueber  das Klebermehl (Bot. Zeit. ,  1855,  p. 
881). — Hartig,  Weitere  Mittheil.  d.  Klebermehl  (Aleuron)  be- 
trefiend  (id.,  1856,  p.  257,  ff.). — Hartig,  Entwicklungsgesch. 
d.  Pflanzenkeims,  Lpz.,  1858,  pp.  108-134. — v.  Holle,  Bei- 
trage  z.  naheren  Kenntniss  d.  Proteinkorner  im  Samen  der 
Gewachse  (Neues  Jahrb.  f.  Pharm.,  Bd.  X,  1858,  pp.  1-24). 
— Cohn,  Ueber  Protein  krystalle  in  den  KartofFeln  (37,  Jahresber. 
d.  schles.  Gesellsch.  f.  vaterl.  Cultur,  1858,  pp.  72-82).— 
Trecul,  Des  formations  vesiculaires  dans  les  cellules  veget. 
(Ann.  des  sc.  nat.  4e  ser.,  t.  X,  1858,  p.  20,  ff.). — Maschke, 
Ueber  d.  Ban  u.  d.  Bestancltheile  der  Kleberblaschen  in  Ber- 
tholletia,  deren  Entwicklung  in  Ricinus  (Bot.  Zeitg.,  1859,  p. 
409,^".). — Radlkofer,  Ueber  Krystalle  proteinartiger  Korper 
pflanzlichen  u.  thierischen  Ursprungs,  Leipz.,  1859. — Nageli, 
Ueber  d.  Krystallahnl.  Proteinkorper  u.  ihre  Yerscheidenh. 
v.  wahren  Krystallen  (Sitzungsber.  d.  K.  Acad.  d.  TViss.  z. 
Miinchen,  Jahrg.,  1862,  Bd.  II,  pp.  120-154).— Cramer,  Das 
Rhodospermin,  ein  krystalloidischer,  quellb;irer  Korper  im 
Zellinhalt  verschiedener  Florideeu  (Vierteljahrsschr.  d.  uatur- 
forsch.  Gesellsch.  in  Ziirich,  Jahrg.,  VII,  1862,  pp.  350-365). 
— Nageli,  Pflanzenphysiol.  Unters. — Sachs,  Zur  Keimungs- 

124  Husemann,  Pflanzenstoffe,  1882,  Bd.  I,  p.  233,  ff. 
125Pfeffer  in  Prin^sheim's  Jahrb.,  Bd.  VIII,  p.  491. 
188  A.  Mayer,  Lehrb.  d.  Agricul.  Chem.,  1877,  p.  191. 


ALBUMINOUS  MATTER.    PROTEIDS.  381 

gesch.  d.  Dattel  (Botan.  Zeitg.,  1862,  p.  241,  jf.).— Sachs, 
Ueber  d.  Keiraung  des  Samens  v.  Allium  cepa  (id.,  1863,  p. 
57,  j^*.). — Gris,  Recherches  anatom.  et  physiol.  sur  la  germi- 
nation (Ann.  des  sc.  nat.  5e  ser.  t.  II,  1864,  pp.  1-123). — 
Cohn,  Beitr.  z.  Physiol.  d.  Phyeochromaceeu  und  Florideen 
(Schultze's  Archiv  f.  mikrosk.  Anat.,  Bd.  Ill,  1867,  pp.  1- 
60.)— Dippel,  D.  Mikrosk.,  Bd.  II,  1869,  p.  29, /".—Klein, 
Ueber  d.  Krystalloide  einiger  Florideen  (Flora,  1871,  pp. 
161-169. — Klein,  Zur  Kentniss  des  Pilobolus  (6  Gefonnte  In- 
haltskorper)  (Pringsheim's  Jahrb.,  Bd.  VIII,  1872,  p.  337,  f.). 
— Pfeffer,  Unters.  iiber  d.  Proteinkorner  u.  d.  Bedeutung  des 
Asparagins  beim  Keimen  der  Samen  (id.,  pp.  419-574). — 
Sachs,  Lehrbuch,  pp.  53-59. — Prillieux,  Sur  la  coloration  et  le 
verdissement  du  Neottia  Nidus-avis  (Ann.  des  sc.  nat.  5e  ser., 
t.  XIX,  1874,  pp.  108-118).— Van  Tieghem,  Nouvelles  re- 
cherches  sur  les  Mucorinees  (Format,  et  role  des  cristalloides  de 
inucorine)  (id.,  6e  ser.,  t.  I,  1875,  pp.  24-32).  —  Schimper, 
Unters.  liber  d.  Proteinkrystalle  d.  Pfl.  Strassb.,  1879. — 
Klein,  Pinguicula  alpina  als  iusectenfr.  Pfl.  u.  in  anatom ischer 
Bezeihung  (Cohn's  Beitrag.  z.  Biol.  d.  Pfl.,  Bd.  Ill,  1880,  pp. 
163-184). — Klein,  Neuere  Daten  iiber  d.  Krystalloide  Meeres- 
algen  (Flora,  1880,  p.  65,  ff.). — Vines,  On  the  Chemical  com- 
pos, of  Aleuron-grains  (Proceedings  of  the  Royal  Soc.  of 
London,  Vol.  XXX,  1880,  p.  387,  f.). 

Proteid  grains  (v.  Holle,  Pfeffer),  also  called  alenron  or  glu- 
ten meal  (Hartig),  were  discovered  in  the  year  1855  by  Theo- 
dore Hartig  and  thoroughly  investigated  first,  by  him,  and  later 
by  Sachs,  but  principally  by  Pfeffer.  They  occur  in  the  endo- 
sperm or  in  the  parenchyma  of  the  cotyledons  of  the  seeds  of  a 
number  of  plants,127  and  indeed, "often  in  company  with  starch, 
embedded  in  the  fatty  protoplasmic  substance  of  the  cells. 
They  are  formed  in  the  last  stages  of  the  ripening  of  the  seeds 
and  change  again  at  the  beginning  of  the  sprouting.  They  are 
represented  by  very  small,  small  or  large  granules.  Sometimes 
there  are  found  in  the  same  cell  many  small  granules  and  one 
great  proteid  grain  (Solitar,  Hartig) .  They  consist  altogether  of 

127  Compare,  concerning  their  occurrence,  Hartig,  Entwicklungsgeschichte  d.  Pflanz- 
keims,  pp.  1-20-124  and  the  above  cited  treatises  of  Pteffer. 


382  THE  MICROSCOPE  IN  BOTANY. 

proteid  matter,  perhaps  being  mingled  with  a  small  quantity  of 
other  substances. 

The  statement  of  Sachs  that  they  consist  in  part  of  fatty 
matter  has  been,  recently,  refuted  by  Pfeffer,  The  mass  of  the 
granules  is  either  amorphous,  or  a  part  of  the  proteid  matter 
assumes  a  crystal-like  nature  (proteid  grains  with  crystalloids). 
Still  others  contain  inorganic  substances.  The  latter  are  either 
true  crystals  of  calcium  oxalate  or  crystalline  roundish  bodies, 
the  so-called  globoids  which  consist  of  calcium  and  magnesium 
phosphates.  We  will,  therefore,  describe  in  their  turn:  (a) 
amorphic  proteid  grains,  (b)  proteid  grains  with  crystalloids, 
(c)  proteid  grains  inclosing  inorganic  matter. 


A.     Amorphic   Proteid  Grains. 

All  amorphic  proteid  grains  are  insoluble  in  absolute  alcohol 
and  ether  (both  must  be  absolutely  anhydrous),  in  chloroform 
and  benzole,  in  fatty  and  essential  oils.  But  benzole  especially 
dissolves  the  fat  of  the  surrounding  fundamental  substance, 
and  in  oily  seeds  the  proteid  grains  appear  more  distinctly 
after  its  application  (for  the  rest  see  below).  If  water  be 
gradually  added  to  a  preparation  lying  in.  alcohol  many  proteid 
grains  show  a  concentric  lamination.  This  appearance  often 
occurs  (Pceonia,  endosperm)  when  alcohol  containing  a  little 
sulphuric  acid  is  employed  and  the  section  examined  in  water.12* 

The  lamination,  however,  appears  only 
in  the  peripheral  part  of  the  grain,  the 
center  remaining  amorphic  (Fig.  134, 
after  Pfeffer)  •  Many  proteid  grains  are 
quite  soluble  in  water,  others  partly, 
still  others  not  at  all.  All  are  soluble 

in  water  which  contains  a  trace  of  caustic  potash,  likewise  in  acids 
and  alkalies,  many  in  glycerine  and  sugar  solution  (in  this  often 
slowly).  Those  grains  which  are  soluble  in  water  must  be  ex- 
amined in  absolute  alcohol  or  oil ;  their  presence  is  best  dem- 
onstrated by  iodine  in  glycerine,  iodine  with  a  little  potassium 
iodide  dissolved  in  glycerine,  see  page  286. 

i28  Pfcffer,  1.  c.,  p.  499. 


ALBUMINOUS  MATTER.    PROTEIDS.  383 

Pfeffer  has  given  a  method  by  which  proteid  grains  which  are 
soluble  in  water  may  be  transformed  into  an  insoluble1  modifica- 
tion. He  says,129  "I  make  use  of  the  property  of  proteid 
matter  to  become  insoluble  in  water  by  corrosive  sublimate  and 
be  transformed  into  an  achlorate  mercurial  combination.  In 
order  to  get  this  without  disorganizing  the  proteid  grain,  I 
digest  the  section  of  the  seed  for  at  least  twelve  hours  in  a  small 
flask  with  a  solution  of  simple  mercuric  chloride  in  absolute 
alcohol,  of  the  concentration  of  which  it  is  not  necessary  to  be 
very  particular,  though  I  find  in  most  cases  about  a  2  per  cent 
solution  to  be  best.  Then  wash  the  section  in  alcohol  and  carry 
it  into  water  in  which  it  is  now  quite  insoluble.  It  is  not  rec- 
ommended to  wash  out  the  section  very  carefully  in  alcohol.  I 
may  also  remark  that  in  taking  the  section  from  the  fluid  no 
needle  or  scalpel  of  steel  should  be  used,  since  iron  makes  a 
precipitate  with  metallic  mercury  which  would  contaminate  the 
surface  of  the  section  coming  in  contact  with  it.  On  this  ac- 
count one  should  use  a  glass  rod  or  most  conveniently  a  needle 
or  small  scalpel  made  of  platinum,  which  latter  one  can  make 
for  himself  for  this  purpose  by  cutting  a  piece  of  thin  platinum 
and  fitting  it  into  a  glass  tube  by  melting  the  glass.  The  pro- 
teid grains  thus  prepared  will  indeed  swell  in  water  but  will 
return  to  their  original  volume  after  a  time  by  treatment  with 
alcohol."  They  are,  however,  soluble  in  dilute  acids  and  al- 
kalies and  give  the  same  reactions  as  the  fresh  proteid  grains. 
The  process  especially  fits  them  for  the  study  of  the  effects  of 
acid  reagents  upon  them. 

By  this  method  of  making  the  proteid  grains  insoluble,  proof 
is  at  the  same  time  afforded  that  gums,  pectinaceous  substances 
or  cane  sugar,  occur,  if  at  all,  only  in  very  small  quantities 
in  proteid  grains.  These  substances  enter  into  no  insoluble 
combinations  with  corrosive  sublimate,  so  that  they  would  be 
dissolved  out  after  this  process  when  subject  to  the  action  of 
water  and  the  granule  would  change  in  form  and  mass  which  it 
does  not  in  fact.130 

If  proteid  grains,  which  are  or  have  been  made  insoluble  in 

«»  Pfeffer,  1.  c.,  p.  141. 
i3o  pfeffer,  1.  c.,  p.  442. 


384  THE  MICROSCOPE  IN  BOTANY. 

water,  be  boiled  with  water  or  treated  with  alcohol  or  ether, 
they  will  coagulate  and  are  then  scarcely  soluble  in  dilute  alka- 
lies and  acids  at  common  temperatures,  but  gradually  dissolve 
in  concentrated  alkali. 

Every  proteid  grain  is  surrounded  with  a  delicate  envelope. 
If  by  the  means  indicated  the  fundamental  substance  of  the 
proteid  grain  be  dissolved  this  envelope  remains  behind  and  the 
application  of  reagents  shows  that  it  consists  of  proteid  sub- 
stances. 

The  principal  microscopical  reactions  of  proteid  grains  are 
the  following.  Iodine  will  in  all  solutions  give  a  brown  or 
yellow-brown  color  (use  a  neutral  solution  where  the  granules 
have  not  been  modified  by  coagulation)  ;  numerous  other  col- 
oring substances  are  also  absorbed  by  the  grains.  Pfeffer131  used 
principally  for  this  purpose  the  aniline  blues  dissolved  in  water, 
solutions  which  remain  unchanged  for  a  long  time.  The  pro- 
teid grains  absorb  therewith,  in  small  quantity,  a  much  more 
striking  color  than  with  cochineal  extract.  Nitric  acid  and 
potash  give  the  yellow  color  proceeding  from  xanthoproteid 
acid ;  sugar  and  sulphuric  acid  a  rose  red  (p.  299)  ;  Millon's  re- 
agent a  brick  red  (p.  296).  The  latter,  according  to  Pfeffer,  is 
not  worthy  of  much  commendation,  likewise  copper  sulphate  and 
potassium  should  be  rejected,  which  Nageli  and  Schwendener132 
have  very  highly  recommended  for  this  purpose.  Cupram- 
monia  does  not  dissolve  the  proteid  granules. 

All  amorphic  proteid  grains  are  not  doubly  refractive  and 
therefore  are  not  illuminated  on  a  dark  field  in  the  polarizing 
apparatus  (Caspary,  Hartig).133 

A  few  words  may  be  added  concerning  the  protoplasmic 
foundation  of  the  cells  which  contain  proteid  grains.134  It  is 
protoplasm  whose  water  is  replaced  by  oil,  the  latter  occurring 
in  the  form  of  small  or  large  drops.  Benzole  or  ether  easily  dis- 
solves it  away  leaving  behind  the  fine,  granular,  often  very  scanty 
fundamental  mass.  The  oily  contents  of  the  fundamental  sub- 
stance is  made  very  beautifully  visible  by  means  of  alcanna  red 

131  Pfeffer,  1.  c.,  p.  444. 

132  Xageli  and  Schwendener,  Mikrosk.,  p.  530. 
«3  Hartig,  Entwicklungsgesch.  d.  Pflk.,  p.  109. 
»«  Pieffer,  I.  c.,  pp.  478-4S5. 


ALBUMINOUS  MATTER.    PROTEIDS.  385 

(Pfeffer).  Use  a  deeply  colored,  about  70  or  80  per  cent,  al- 
coholic extract  of  alcanna  root  (see  p.  310).  Move  the  seed- 
section  back  and  forth  in  this  a  few  times,  wash  it  off  in  weak 
alcohol,  and  put  it  immediately  in  strong  glycerine  for  examin- 
ation. The  coloring  matter  dissolved  in  alcohol  does  not  pene- 
trate into  the  proteid  granules  in  the  short  space  of  time  in  which 
it  comes  in  contact  with  them.  But  the  alcoholic  solution  of 
the  coloring  matter  adhering  to  the  section,  is  quickly  dissolved 
by  the  oil  in  the  fundamental  mass  with  which  it  comes  into 
such  intimate  contact..  By  its  distribution  through  the  funda- 
mental mass,  sections  of  very  oily  seeds  are  soon  colored  a  beau- 
tiful blood  red.  When  the  fundamental  substance  itself  is  very 
deeply  colored  the  proteid  grains  appear  to  be  isolated,135  quite 
colorless.  If  the  oil  is  very  easily  dissolved  in  alcohol,  as  in 
the  seeds  of  Hicinus,  the  alcanna  tincture  should  be  diluted  with 
an  equal  quantity  of  glycerine. 

If  the  cells  containing  proteid  grains  be  treated  with  potash 
solution  or  potash  water  after  the  oil  has  been  removed  with 
benzole  or  ether,  the  grains  will  be  dissolved  away  and  the 
fundamental  mass,  as  well  as  the  envelopes  of  the  granules,  will 
,be  left  as  a  delicate  network,  which  looks  often  not  unlike 
parenchyma  tissues.136  Iodine  solutions  color  it  brown-yellow, 
Millon's  reagent  red.  It  consists  of  albuminous  matter.  The 
nucleus  lies  shrunken  up  within. 


B.     Proteid  Grains  with  Crystalloids. 


187 


A  great  number  of  proteid  grains  contain  formed  proteid 
matter,  which  appears  in  the  form  of  crystal-like  bodies.  Har- 
tig  called  them  "white  granules,"  Nageli  named  them  crystal- 
loids. They  are  surrounded  by  the  envelope  of  the  granule 
(Fig.  135,  A,  B,) ,  which  may  moreover  often  be  almost  entirely 
wanting.  Sometimes  several  crystalloids  are  found  united  in  one 
grain  (Fig.  135,  C,  D,  after  Pfeffer).  In  oil  and  alcohol  the 
crystalloids  are  not  usually  visible  in  consequence  of  the  like 

IBS  pfeffer,  1.  c.,  Taf.  XXXVIII,  Fig.  21. 
no  Pfeffer,  L  c,,  Taf.  XXXVIII,  Fig.  2. 
«7  Pfeffer,  I.  c.,  pp.  450-iGi. 


386  THE  MICROSCOPE  IN  BOTANY. 

refracting  power  of  the  crystalloids  and  the  envelope.  In  order 
to  make  the  former  visible  the  grains  must  be  put  into  water. 
This  either  dissolves  the  surrounding  mass  of  the  envelope  or 
makes  it  swell  thus  causing  the  crystalloids  to  appear.  The 
enveloping  mass  has  the  same  qualities  as  the  substance  of  the 
proteid  grains  without  crystalloids. 

Of  the  characteristics  of  the  crystalloids  we  mention  the  fol- 
lowing :    The  crystal  systems  are  not  very  thoroughly  known. 
The  crystalloids  of  Bertholletia  should,  according  to  Nageli,  be 
clinorhombic  ;  others  belong,  according  to  Hartig,  to  the  tesseral 
and  rhombic  systems.     Sorauer  found, 
besides  these,  four-sided  columns.     All 
crystalloids,  although  but  slightly,  are 
double  refractive.138  According  to  Radl- 
koferthis  latter  characteristic  is  increased 
by   coagulation.       The   angles    of    the 
crystalloids  are  very  inconstant ;  the  ad- 
dition of  water  changes    them    2    or   3 
degrees,  swelling  about  15  or  16  degrees. 
According  to  Nageli  the  crystalloids,  like 
the  starch  grains,  consist  of  two  distinct 

substances  (see  p.  360),  according  to  Maschke,  of  casein  and  a 
little  albumen  ;  according  to  PfefFer  both  views  are  groundless. 
All  crystalloids  are  insoluble  in  water ;  they  can,  however,  be 
freed  from  the  fundamental  mass  by  water  alone  or  with  the 
addition  of  sodium  phosphate.  They  are  insoluble  likewise  in 
absolute  alcohol  but,  on  the  contrary,  are  soluble  in  glycerine 
containing  potash,  in  dilute  potash  as  well  as  in  muriatic  and 
acetic  acid.  After  solution  every  crystalloid  leaves  behind  a 
little  envelope  (Fig.  135,  E  and  F,  after  Pfefier).  The  crys- 
talloid should  be  dissolved  by  concentrated  glycerine  with  a 
trace  of  potash.  The  envelope  gives  the  same  reaction  as  that  of 
the  grain  itself.  By  boiling,  the  crystalloids  are  transformed 
into  the  insoluble  modification  of  proteid  matter.  They  are 
then  insoluble  in  dilute  potash  but  will  swell  considerably  in  it. 


»8  Maschke  in  Bot.  Zeitg.,  1839,  Tab.  XV,  Figs.  95,  96,  98,  99, 101,  102. 


ALBUMINOUS  MATTER.     PROTEIDS.  387 


Proteid  Crystalloids  without  an  Inclosing  Mass. 

In  the  proteid  grains  containing  crystalloids  the  inclosing 
mass  is  often  quantitatively  considerable,  but  sometimes  the 
crystalloids  are  inclosed  only  by  a  very  thin  layer.  These  pro- 
teid grains  are  closely  related  to  those  crystalloids  which  are 
quite  uncovered.  In  the  latter  case  the  proteid  crystalloids  lie 
free  in  the  cells  or  in  the  plasma.  A  sharply  defined  boundary 
between  the  free  lying  crystalloids  and  those  inclosed  in  an 
integumentary  mass  can  scarcely  be  drawn.  Free  proteid  crys- 
talloids frequently  occur  in  resting  seeds  as  well  as  elsewhere,  in 
phanerogams  as  well  as  in  cryptogams.  We  limit  ourselves 
here  to  the  presentation  of  some  carefully  studied  cases. 

1.  Crystalloids  of  the  Potato  tuber  (Cohn).     In  the  cuticular 
layer   of  the  potato    tuber   occur  numerous  cubic  crystalloids 
measuring  from  0.007  to  0.013  mm.  on  a  side.     Cohn139  gives 
the  following  reactions  for  these  forms  which  are  feebly  double 
refractive.      Iodine  colors  them  yellow  to  deep  golden  browny 
sugar  and  sulphuric  acid  a  peach  blow  red,  Millon's  reagent  a 
brick    red,    carmine  with    the  least  possible  ammonia  a  dee[> 
red,  cochineal  extract  with  water  and  a  subsequent   addition 
of  acetic  acid  (Maschke)  intense  burning  red.     They  are  slowly 
soluble  in  glycerine,  soluble  in  ammonia  from  outward  toward 
the  center,  in  acetic  acid  from  the  center  outward,  and  in  dilute 
potassium  lye  (but  not  in  concentrated ;  in  this  they  swell  and 
are  stained  yellow) .     Sulphuric,  nitric  and  muriatic  acid  dissolve 
most  of  the  crystalloids  or  make  them  swell  up  to  a  globular* 
drop.     By  boiling  in  water  the  crystalloids  in  general  remain 
unchanged  but  become  more  easily  visible  and  show  a  laminated 
structure. 

2.  Crystalloids  in  Bertholletia   excelsa    (Hartig,  Maschke, 
Radlkofer,  Nageli) .     The  crystalloids  in  the  endosperm  of  the 
Para  nut  have  been  frequently  described,  but  the  statements  con- 
cerning the  reactions  produced  on  them  by  the  several  naturalists 
are  wide  apart,  which  Nageli  claims  is  chiefly  caused  by  using 
the  reagents  in  very  different  degrees  of  concentration  by  which 

"9  Colin,  58  Ber.  d.  Schlea.  Gesellsch.  f.  vaterl.  Cultur,  1859,  pp.  74-77. 


388 


THE  MICROSCOPE  IN  BOTANY. 


the  tested  crystalloids  would  present  themselves  in  a  very  dif- 
ferent manner.  Hartig  held  the  crystalloids  to  be  hexagonal, 
Maschke  tesseral ;  Nageli  finally  made  it  very  probable  that  they 
are  clinorhombic  (Fig.  136,  A,  B,  after  Nageli).  According 
to  Nageli  they  do  not  dissolve  in  water.  Hartig  had  stated  that 
they  were  soluble  in  water,  Radlkofer  that  it  took  but  slight 
hold  of  them.  By  boiling  in  water  they  coagulate  and  are  not 
then  soluble  in  weak  alkali  (Radlkofer).  Alcohol  and  ether  do 
not  alter  them  even  when  boiling  hot.  According  to  Nageli 


FIG.  136. 

glycerine  does  not  dissolve  them,  only  increases  their  volume. 
JRadlkofer  says  that  they  dissolve  in  it  very  slowly.  Acetic 
acid  will  not  dissolve  them  even  in  the  presence  of  glycerine. 
Very  weak  acids  do  not  change  the  crystalloids,  stronger  dissolve 
them  gradually  or  quickly  ;  first,  however,  making  them  swell  up 
,(Fig.  136,  C,  D).  In  nitric  acid  they  become  round  and  full  of 
vacuoles  and  in  time  yellow.  Ammonia  dissolves  them  with 
.less  swelling,  likewise  weak  potash,  while  the  concentrated  does 
not  dissolve  them,  only  rounds  them  up.  Iodine  colors  them 
brown  or  yellow  brown,  Millon's  reagent  red  ;  coloring  matter 
is  energetically  absorbed  by  them.140 

3.  Crystalloids  in  Pilobolus  (Klein,  Van  Tieghem),  In  the 
fruit  bearers  of  Pilobolus  occur  likewise 
many  small  crystalloids  which  were  in- 
vestigated in  Pilobolus  crystallinus  by 

/$i     IA        \]  ^^    Klein,  and  in  P.  roridus  by  Van  Tie- 
ghem    (Fig.   137,  after  Van  Tieghem 
FIG.  137.  and  Klein).     They  are  colorless    and 

appear  to  be  octahedral  or  quadratic  pyramidal,  and  have  sides 

Radlkofer,  Krya- 


Nageli,  Sitzungsber.  Bayer.  Acad.,  1862,  Bd.  II,  pp.  128-13 
talle  proteiuartiger  Korper,  pp.  65-69. 


ALBUMINOUS  MATTER.     PROTEIDS.  389 

which  are  not  quite  plane.  Potash  either  swells  or  dissolves 
them.  Iodine  colors  them  yellow  or  brown.  If  the  iodine  is 
dissolved  in  alcohol  they  are  shrunken  at  the  same  time  ;  alcohol 
alone  contracts  them.  Sulphuric  acid  alone  colors  them  a  rose 
red.  It  colors  the  plasma  of  Pilobolus  likewise  the  same.  The 
long  continued  action  of  concentrated  nitric  acid  colors  them  a 
pale  yellow.141 

4.  Crystalloids  in  Floridia.  (Rhodospermin.)  (Cramer, 
Klein,  Cohn) .  Crystalloids  of  proteid  bodies  have  been  observed 
in  different  algae,  as,  for  example,  Bornetia  secundiflora  Thuret, 
Callithammon  caudatum  Ag.,  C.  seminudum  Ag.,  Griffithsia 
barbata  Ag.,  Gr.  Nepolitana  Nag.,  Gongocerus  pellucidum 
Ktzg.,  and  designated  by  Cramer  Rhodospermin,  since  they 
have  commonly  been  stained  by  the  red  coloring  matter  of  the 
algae  and  are  rose  red.  Cohn  and  Klein  demonstrated  afterwards 
that  the  crystalloids  in  the  living  plants  are  mostly  colorless. 
The  crystalloids  are  either  simply  refractive  and  belong  then  to 
the  hexagonal  system  (needles  or  plates  of  from  0.004  to  0.050 
mm.  long),  or  they  are  double  refractive,  clinorhombic  (octa- 
hedral-like  forms  of  from  0.01  to  0.04  mm.  long).  Cramer 
distinguished  therefore  hexagonal  and  clinorhombic  Rhodosper- 
rnin.  (a)  Hexagonal  Rhodospermin:  insoluble  in  water,  and 
absolute  alcohol  (cold  or  boiling) ,  glycerine  and  acetic  acid,  cold 
muriatic  acid  (boiling  slowly  destroys  it),  sulphuric  acid  like- 
wise and  nitric  acid.  Nitric  acid  alone  does  not  color  it,  but 
does  with  the  addition  of  ammonia.  It  is  insoluble  in  dilute  and 
concentrated  potash  lye,  ammonia  and  cuprammonia.  These 
substances,  however,  cause  it  to  swell  and  in  boiling  potash  lye 
it  is  slowly  destroyed.  Iodine  colors  it  first  gold-yellow  then 
brown-yellow ;  ammoniacal  carmine  solution  colors  it  red  but 
not  essentially  different  from  the  surrounding  solution  ;  carmine 
solution  with  the  addition  of  cooking  salt,  on  the  contrary,  an 
intense  red.  Sugar  with  sulphuric  acid  gives  no  reaction,  (b) 
Clinorhombic  Rhodospermin:  Iodine,  nitric  acid  with  ammonia 
behave  as  with  the  hexagonal  Rhodospermin.  In  potash  and 

141  Klein  in  Pringsheim's  Jahrb.,  Bd.  VIII,  p.  337.  _/F.,uncl  376.— Van  Tieghemiu  Ann.  des 
sc.  nai.  6e  Ser.,  t.  I,  p.  25,/. 


390  THE  MICROSCOPE  IN  BOTANY. 

ammonia  it  swells  but  contracts  again  with  nitric,  sulphuric  or 
muriatic  acid.  Millon's  reagent  colors  it  a  brownish  yellow.142 
5.  Colored  Crystalloids  in  the  pulp  of  the  fruit  of  Solatium 
Americanum  Mill  (Nageli).  These  occur  in  the  form  of  thin 
plates  and  rhombs  (Fig.  138,  A,  after  Nageli)  which  are  fre- 
quently united  (B),  belong  to  the  rhombic  system  and  possess 
an  intense  violet  color.  The  crystalloids  consist  partly  of  albu- 


FlG.  138. 

minous  substances  which  are  permeated  with  the  violet  coloring 
matter.  Water  does  not  alter  them  but  if  it  be  slightly  sour  or 
alkaline  the  tone  of  the  color  is  changed.  Alcohol  bleaches 
them  from  the  inside  outward  and  dissolves  most  of  them, 
likewise  ether.  Iodine  colors  them  a  brown-yellow.  Very 
weak  acids  color  the  crystalloids  bright  red,  strong  acids  bleach 
and  disintegrate  them  into  separate  pieces  and  finally  dissolve 
them.  Potash  and  boiling  water  behave  alike;  essential  oils 
and  chloroform  are  without  effect  upon  the  dry  crystalloids.143 

C.     Proteid  grains  inclosing  Inorganic  Substances. 

As  already  briefly  mentioned  there  are  proteid  grains  which 
contain  within  themselves  inorganic  substances.  These  are 
either  globoids,  spheroids  (Fig.  139,  A,  after  Pfeffer),  or 
crystals  (B).  The  former  are  a  combination  of  magnesia  and 
lime  with  a  little  phosphoric  acid,  the  latter  consists  of  calcium 
acetate.  Frequently  spheroids  and  globoids  occur  with  crystal - 

142  According  to  Cramer,  I.  c.,  Cohn  in  Schultze's  Archiv,  Bd.  Ill,  p.  24,  /.    Klein  in 
Flora,  1871,  pp.  161-169. 

143  Nageli,  Sitzungsber.  d.  Bayer.  Acad.,  1862,  Bd.  II,  pp.  147-154;  Vgl.  auch  Nageli  in 
Pflanzenphysiol.  Uiiters.,  Bd.  I,  p.  6. 


ALBUMINOUS  MATTER.    PROTEIDS.  391 

loids  iu  the  same  proteid  grains,  but  more  seldom  globoids  with 
crystals. 

(a)  Proteid  grains  with   globoids.      The  globoids  (Kranz- 
korper  of  Hartig)  have  a  roundish  or  cluster-like  form  (Fig. 
139,  A) .     They  occur  in  almost  every  seed  that,  contains  reserve 
proteid  matter.     The  largest  (Vitis)  attain  a  diameter  of  0.01 
mm.     In  order  to  investigate  globoids  (as  crystals)  first  remove 
the  oil  from  the  section  of  the  seed  and  then  dissolve  away  the 
proteid  substance  with  water  or  very 

dilute  potash  (Pfeffer) .  The  globoids 
are  singly  refractive,  insoluble  in  cold 
and  boiling  water  and  alcohol,  soluble 
in  all  mineral  acids  and  acetic  acid 
(without  effervescence).  They  take 
no  color  from  iodine  or  aniline  blue. 
They  gradually  dissolve  in  an  ammo- 
niacal  chlorine-ammonia  solution,  like- 
wise in  alcohol  which  contains  a  little  sulphuric  or  oxalic 
acid.  In  the  latter  case  after  a  considerable  time  one  may 
find  in  place  of  the  globoids  tiny  needle  crystals  of  calcium 
and  magnesium  oxalate.  Concentrated  potassium  and  am- 
monia dissolve  a  substance  out  of  the  globoids  from  the  outside 
inward.  They  then  appear  as  a  finely  granulated,  feebly  re- 
fractive mass  with  a  cuticular  layer  which  may  be  stained  like 
proteid  matter  with  iodine  and  aniline  blue.  No  swelling  is 
produced  by  the  action  of  the  potash. 

(b)  Proteid   grains  with    crystals.      The  crystals  occur  as 
clinorhombic  plates,  etc.,  or  as  a  cluster  of  crystals  grown  to- 
gether.    They  are  insoluble  in  water,  and  acetic  acid  ;  calcined, 
the  residuum  dissolves  in  the  latter  with  effervescence.     They 
are  insoluble  in   not  too  concentrated  potash  lye.      (For  fur- 
ther reactions  see  under  section  XII,  Inorganic  Vegetable  Ele- 
ments.)    If  the  crystals   are  carefully   dissolved  in   muriatic 
acid,  there   remains  behind  a  delicate  skin  consisting  of  proteid 
matter,  also  in  the  middle  something  like  a  nucleus  is  found. 
Both  can  be  recognized  with  certainty  when  a  little  iodine  is 
added  to  the  dilute  muriatic  acid  used  in  the  solution. 


392  THE  MICROSCOPE  IN  BOTANY. 

2.     FUNCTIONAL  PROTEID  MATTER. 
(Protoplasm  and  Cell  nucleus.) 

Literature.  V.  Mohl,  Einige  Bemerk.  iiber  d.  Bau  d.  veget. 
Zelle  (Bot.  Ztg.,  1844,  p.  273, /".).— V.  Mohl,  Verm.  Schr. 
Tubing.,  1845,  a.  v.  O.— V.  Mohl,  D.  Veget.  Zelle,  Brschwg., 
1851,  p.  198,  ff. — Schacht,  D.  Pflanzenzelle,  Leipzig,  1852,  a. 
v.  O. — Hiirtig,  Ueber  d.  Verf.  b.  Behandl.  d.  Zellkerns  mit  Farb- 
stoffen  (Bot.  Zeitg.,  1854,  p.  877,  ff.).— v.  Mohl,  D.  Primordi- 
alschlanch  (Bot.  Zeitg.,  1855,  p.  689, /.).— Schacht,  Lehrb.  d. 
Anat.  u.  Physiol.  d.  Gew.,  1856,  a.  v.  O.  —  Hartig,  Eutwick- 
lungsgeschicte  d.  Ptikeims,  Lpz.,  1858,  a.  v.  O. — Maschke,  Pig- 
mentlosung  als  Reagenz  bei  mikrosk.  physiol.  Unters.  (Botan. 
Zeitg.,  1859,  p.  21,  ff.). — Raxllkofer,  Ueber  Krystalle  proteinar- 
tiger  Korper,  Lpz.,  1859,  p.  1,  ff. — Sachs,  Ueber  einige  neue 
mikrosk.-chem.  Reactionsmeth.  (Sitzungsber.  d.  K.  Acad.  d. 
Wiss.  Wieu,  Bd.  XXXVI,  1859,  p.  9,  f.).—Ve  Bary,  Ueber  d. 
Bauu.  d.  Wesender Zelle  (Flora,  1862,  pp.  243-251).— Sachs, 
Mikrochem.  Unters.  (Flora,  1862,  p.  297, /".).— Schultze,  Ue- 
ber d.  Bau  d.  Nasenschleimhaut  (Abh.  d.  naturf.  Gesellsch.  zu 
Halle,  Bd.  VII,  1863,  p.  92). — Sachs,  Zur  Keimungsgesch.  d. 
Graser  (Bot.  Zeitg.,  1862,  p.  145,  ff.). —  Sachs,  Zur  Keimungs- 
gesch. d.  Dattel  (id.,  p.  241,  ff.).—  Schultze,  D.  Protoplasma 
d.  Rhizopoden  u.  d.  Pflanzenzellen,  Lpz.,  1863,  p.  39,  ff.).— 
Cienkowsky,  Zur  Eutwicklungsgesch.  d.  Myxomyceten  (Pring- 
sheim's  Jahrb.,  Bd.  Ill,  1863,  p.  325,  ff.).— Cienkowsky,  D. 
Plasmodium  (id.,  p.  400,  ff.). —  Sachs,  Beitrage  z.  Physiblog. 
d.  Chlorophylls  (Flora,  1863,  p.  193,  ff.).— Sachs,  Ueber  d. 
Keimung  d.  Samens  von  Allium  cepa  (Bot.  Zeitg.,  1863,  p. 
57,^".). — Sachs,  Ueber  d.  Stoffe,  welched.  Material  z.  Aufbau 
d.  Zellhaute  liefern  (Pringsheim's  Jahrbuch,  Bd.  Ill,  1863, 
p.  185,  ff.).—  De  Bary,  D.  Mycetozoen,  Leipz.,  1864,  p.  41, 
ff. — Kiihne,  Unters.  iiber  d.  Protoplasma,  Leipz.,  1864. — 
Sachs,  Handbuch  d.  Experimentalphysiol.  d.  PfL,  Leipz.,  1865, 
p.  309,  ff.— De  Bary,  Morph.  u.  Physiol.  d.  Pilze,  Flechten 
u.  Myxomyc.,  Leipz,.,  1866,  p.  103,  /. — Nageli  u.  Schwen- 


FUNCTIONAL  PROTEID  MATTER.  393 

dener,  Mikrosk.,  p.  529,  ff. — Hanstein,  Ueber  d.  Organe  d. 
Harz-  u.  Schleimabs.  an  d.  Laubknospen  (Botan.  Zeitg.,  1868, 
p.  697,  ff.).—  Dippel,  Mikrosk.,  Bd.  II,  Brschwg.,  1869,  pp. 
9-18.  —  Schroder,  Beitr.  z.  Kenntn.  der  Friihjahrsperiode 
des  Ahorn  (Pringsheim's  Jahrb.,  Bd.  YII,  1869,  pp.  283, 
314,  325). — Sachs,  Lehrb.,  p.  39,  ff. — Strasburger,  Stu- 
dien  iiber  Protoplasma.  Jena,  1876. — Tangl,  D.  Protoplasm* 
d.  Erbse  (Sitzungsber.  d.  K.  Acad.  d.  AViss.  Wieu,  Bd. 
LXXVI,  1877,  Decemberheft,  Bd.  LXXVIII,  1878,  Juniheft.) 
— Treub,  Quelques  rech.  sur  le  role  du  noyau  dans  la  divis.  des 
cellules  veget.,  Amsterd.,  1878. — Behrens,  D.  Xect.  d.  Bliiten 
(Flora,  1879,  a.  v.  O.) — Schmitz,  Unters.  iiber  Structur  d. 
Protopl.  u.  d.  Zellkerne  d.  Pflzellen  (Sitzungsber.  d.  uiederrh. 
Gesellsch.  zu  Bonn,  1880,  p.  159,  ff.)—  Strasburger,  Zellbild- 
ung  u.Zelltheilung,  Jena,  1880,  a.  v.  O. — Johow,  Uuters.  iiber 
d.  Zellkem  der  hoheren  Monokot.  Bonn,  1880. — Hanstein,  D. 
Protopl.  als  Trager  d.  pflanzl.  u.  thier  Lebensverricht.  Heid- 
elbg.,  1880. — Reinke,  Studien  iiber  Protoplasma,  Berlin,  1881. 
— Tangl,  Ueber  offeue  Communu.  zwischeu  d.  Zellen  d.  Endosp. 
einiger  Sameu  (Pringsheim's  Jahrb.,  Bd.  XII,  1881,  p.  170, 
ff.). — Detmer,  D.  Wesen  d.  Stoffwechselprocesse  im  veg.  Or- 
ganismus  (id.,  p.  253,  ff.).  Poulsen,  Botan.  Mikrochem.,  p. 
52,  /.  (Trans,  p.  63)144  [E.  Pfitzer  in  Bericht  Deutsch.  Botan. 
Gesellsch.  I  (1883),  pp.  44-77.] 

Functional  proteid  substances,  protoplasm  and  cell  nucleus 
are  found  in  all  living  cells,  and  on  account  of  their  constant 
occurrence  are  also  generally  known.  Functional  proteid  sub- 
stance either  fills  the  cell  and  has  no  constant  form  as  a  whole 
(protoplasm),  or  it  has  a  form  and  is  localized  (lying  in  the 
protoplasm)  and  is  enclosed  in  a  delicate  cuticular  membrane 
(cell  nucleus).  Protoplasm  is  not  always  enclosed  in  the  cellu- 
lose walls  of  surrounding  cells  ;  it  may  also  exist  in  a  living 
form  by  itself  (Amoeba,  Plasmodia,  Myxomyceta,  swarm 
spores,  etc.)  It  frequently  exhibits  (free  or  in  cells)  charac- 
teristic appearances  of  motion.  It  is  seldom  represented  by  a 

144  A  perfect  list  of  the  literature  concerning  this  subject  is  really  impossible.  In  the 
present  list  only  those  treatises  are  quoted  which  describe  microscopical  methods  of  re- 
action. All  others,  for  instance  those  which  treat  of  the  appearances  of  the  movement  of 
protoplasm,  are  not  referred  to. 


394  THE  MICROSCOPE  IN  BOTANY. 

horny,  hard  mass  as  in  resting  seeds.  In  most  cases  it  is  per- 
meated by  a  greater  or  less  quantity  of  water  and  then  is  plastic, 
soft  and  often  very  like  a  fluid.  In  it  are  almost  always  small  or 
very  small  granules  (oil  drops)  by  which  it  gets  a  granulated  ap- 
pearance. The  protoplasmic  body  is  commonly  surrounded  out- 
wardly by  a  solid  hyaline  ungranulated  layer  (cuticular  layer). 
Protoplasm  is  composed  chemically,  first  of  all,  of  the  (frequent- 
ly prevailing)  albuminous  substances,  also  of  a  great  number  of 
other  combinations  (Sachs,  Reinkeand  Rodewald)  ;  and  lastly, 
of  a  small  quantity  of  inorganic  incombustible  substances. 

There  are  often  found,  temporarily  or  otherwise,  in  the  proto- 
plasm, other  substances  which  are  afterwards  employed  either 
for  building  the  wall  of  the  cell  (cellulose  builder,  Sachs), 
or  which  are  separated  out  as  substances  of  secretion  (meta- 
plasm,  Hanstein).  The  nucleus,  which,  as  has  been  established 
by  the  recent  investigations  of  Strasburger,  Hanstein,  Treub, 
Schultz  and  others,  plays  an  important  part  in  cell  division, 
consists  of  several  elements,  concerning  which  one  must  consult 
the  authors  named. 

All  reactions  to  be  hereafter  referred  to  show  both  in  the 
protoplasm  and  the  nucleus,  since  in  both  albuminous  sub- 
stances predominate.  Both  will  be  described  separately,  care 
being  taken  that  in  treating  of  the  nucleus,  the  methods  already 
given  with  reference  to  protoplasm  be  not  repeated. 

A.     Protoplasm,  Epiplasm,  Metaplasm. 

1.  Protoplasm  in  the  narrower  sense.  Substances  which 
absorb  water,  as  absolute  alcohol,  concentrated  glycerine,  solu- 
tion of  common  salt,  absorb  the  water  from  protoplasm  and 
cause  it  to  shrink  up  or  contract.  It  thus  draws  itself  away 
from  the  cell  wall  and  commonly  assumes  an  irregular  outline. 
Absolute  alcohol,  osmic  acid,  solution  of  picric  acid  kill  proto- 
plasm and  cause  it  to  stiffen.  The  more  quickly  this  stiffening 
or  hardening  takes  place,  as,  for  example,  in  boiling  alcohol,  the 
more  perfectly  is  the  original  structure  preserved  (Strasburger, 
see  p.  178).  A  solution  of  common  salt  does  not  kill  the 
shrunken  protoplasm.  A  cell  so  treated  is  "  plasniolized." 


FUNCTIONAL  PROTEID  MATTER.  395 

Plasma  contracted  by  means  of  alcohol  appears  to  be  far  less 
soluble  in  acids  and  dilute  alkalies  than  when  fresh  (v.  Mohl). 

A  characteristic  quality  of  dead  protoplasm  is  its  ability  to 
absorb  a  large  number  of  coloring  substances.  Living  proto- 
plasm does  not  possess  this  power  as  already  proved  by  Hartig, 
who  grew  Algce,  Lemma,  Cham,  Hydrocharis  in  carmine  so- 
lution. The  growth  was  not  particularly  hindered  by  the  col- 
oring matter,  but  the  protoplasm  and  nucleus  absorbed  not  the 
least  trace  of  the  pigment.145  Furthermore  the  nucleus  has  the 
power  of  absorbing  the  coloring  matter  in  a  much  higher  degree 
than  the  protoplasm.  Grenadier's  carmine  solution  (p.  308) 
and  most  of  the  other  carmine  solutions  as  well  as  the  extract 
of  cochineal  (see  p.  305)  can  be  commended.  The  resistant 
enveloping  layer  of  the  protoplasm  behaves  quite  negatively 
towards  most  coloring  substances.146  Hanstein's  aniline  solution 
will  be  taken  up  by  unchanged  protoplasm  as  a  blue-violet. 
Iodine  in  the  familiar  solutions  (potassium  iodide  of  iodine, 
chlor-iodide  of  zinc,  glycerine  iodine,  and  iodine  and  sulphuric 
acid),  will  give  a  yellow  or  brown-toned  color.  The  brown 
shades  are  the  most  common  and  in  many  cases  are  very  dark. 
,  The  alkalies  behave  toward  protoplasm  differently  according 
to  their  degree  of  concentration,  those  most  in  use  being  potash 
and  soda  lyes  whose  action  is  much  like  that  of  the  rest.  Con- 
centrated solution  of  potash  leaves  protoplasm  entirely  un- 
changed, neither  dissolving  nor  swelling  it.  M.  Schultze147 
therefore  recommends  strong  potash  lye  as  a  mounting  medium 
for  protoplasmic  preparations.  Dilute  potash  solutions  on  the 
contrary  make  protoplasm  first  transparent  and  then  soon  per- 
fectly dissolves  it.  Concentrated  ammonia  fluid  clears  it  up 
very  soon  and  dissolves  it,  though  sometimes  not  perfectly  till 
after  several  hours. 

Concentrated  mineral  acids,  for  example,  concentrated  sul- 
phuric acid,  have  the  greatest  dissolving  power.  This  acid  does 
not  commonly  color  protoplasm,  but  if  the  protoplasm  be  anhy- 
drous it  becomes  rose-red  to  brown.  Sulphuric  acid  with  con- 

»"  Hartig  in  Bot.  Zeitg.,  1854,  pp.  576,  877. 

i46Tangl  in  Pringsheim's  Jahrb.,  Bd.  XII,  p.  174. 

*47  M.  Schultze  in  Abhandl.  d.  naturf.  Gesellsch.  z.  Halle,  Bd.  VII,  p.  92,  f. 


396  THE  MICROSCOPE  IN  BOTANY. 

centrated  solution  of  sugar  colors  every  kind  of  protoplasm 
rose-red,  and  moreover  this  reagent  is  quite  sensitive.  First 
lay  the  preparation  in  the  sugar  solution,  then  put  on  a  cover- 
glass  and  let  the  acid  flow  in  from  the  edge.  Phosphoric 
acid  changes  protoplasm  but  little  ;  acetic. acid  makes  it  opaque. 
Nitric  acid,  used  warm  or  cold,  colors  it  yellow  or  brown  with 
the  formation  of  xanthoproteid  acid ;  by  adding  potassium  or 
ammonia  there  will  be  formed  the  related  xanthoproteid  salt  which 
is  distinguished  by  its  positive,  mostly  brown,  color. 

Millon's  reagent  colors  protoplasm  brick-red,  still  it  is  on  the 
whole  not  very  sensitive.  Indol  with  sulphuric  acid  colors  it  a 
feeble  rose-red  if  at  all  (Niggl). 

Copper  sulphate  with  potash  (method  p.  365)  colors  all 
albuminous  substances  violet,  which  color  will  not  be  changed 
by  continued  boiling.  By  reflected  light  it  is  uniformly  a  dark 
violet ;  by  transmitted  it  plays  more  into  the  wine-red.148 

The  Plasmodium  of  the  Myxomycetc^®  becomes  rose-red  by 
sugar  and  sulphuric  acid,  and  Millon's  reagent  with  iodine  yel- 
low. Alcohol  and  nitric  acid  cause  coagulation ;  in  acetic  acid 
the  substance  becomes  colorless  and  transparent.  It  liquefies 
in  dilute  potash  solution,  likewise  in  potassium  carbonate  which 
first  often  somewhat  shrinks  it.  Alcohol,  glycerine,  chlorate  of 
zinc,  iodine  and  dilute  chromic  acid  leave  the  marginal  layer  at 
first  unchanged,  but  on  the  other  hand  quickly  contracts  the 
remainder  of  the  plasmodium. 

2.  Epiplasm.      De    Bary150   designates    by   this   name    the 
protoplasmic   residuum  in    the    spore  sacs  of  the   Ascomycetm 
which  still  remains  after  the  spores  are  formed.       It  is  more 
strongly  refractive  than  the  common  protoplasm,  has  a  charac- 
teristic, homogeneous  sparkling  appearance,  and  is  very  sensi- 
tive toward  iodine  solutions,  the  most  dilute  of  which  colors  it 
a  beautiful  red  to  a  violet-brown. 

3.  Metaplasm.     Under  this  designation  of  Hanstein151  we 
are   to   understand   protoplasm    in   which   are    contained  con- 
siderable  quantities    of    carbo-hydrates,    the   most    important 

"8  Sachs  in  Sitzungsber.  d.  K.  Acad.  d.  Wiss.  Wien,  Bd.  XXXVI,  p.  9. 

"9  De  Bary,  d.  Mycetozoen,  p.  41,  /. 

is"  De  Bary,  Morphology  und  Physiol.  d.  Pilz.  Flectin  und  Myxonwc.,  p.  103,  /. 

«i  Hanstein  in  Bot.  Zeit.,  1868,  p.  710. 


FUNCTIONAL  PROTEID  MATTER.  397 

of  which  are  the  amyloid-like  substances,  which  sooner  or 
Liter  will  be  separated  from  it  to  be  applied  to  the  construc- 
tion of  cell  walls  or  as  secretions.  In  many  organs  of  secretion 
the  albuminous  substances  of  the  metaplasm  are  so  far  thrust 
into  the  background  that  it  is  with  the  greatest  difficulty  they 
are  detected  by  the  reagents  commonly  used.152  In  the  remain- 
der the  albuminous  substances  are  detectable  by  the  previously 
described  methods  of  reaction.  Metaplasm  behaves  character- 
istically toward  Hanstein's  aniline  mixture  as  has  already  been 
shown.  It  is  not  colored  by  it,  like  common  protoplasm,  blue- 
violet153,  but  scarlet-red,  this  color  being  more  fuchsin-red  when 
tannic  acid  is  present  in  it  (see  below). 

B.     Cell  Nucleus. 

As  has  been  previously  indicated,  the  cell  nucleus  gives  the 
same  reactions  as  other  proteid*substances.  For  a  long  time  a 
considerable  number  of  histological  reagents  have  been  employed 
to  make  the  nucleus  itself  more  clear  and  bring  out  all  the 
fine  structural  relations  which  had  heretofore  been  indistinct. 
These  reagents  are  again  in  use,  and  especially  since  the  epoch- 
%making  investigations  of  Strasburger.  The  unusual  activity  in 
the  study  of  the  nucleus  has  brought  to  light  a  great  number 
of  these  reagents.  WQ  can,  therefore,  in  the  following,  name 
but  a  fe\v  of  the  more  important,  and  for  the  rest  may  refer 
to  those  works  which  treat  of  these  matters,  and  which  every 
phytotomist  who  would  be  conversant  with  the  questions  of  the 
day  must  study. 

Hartig  first  attempted  to  stain  the  nucleus  with  a  carmine 
solution  in  water  which  had  absorbed  ammonia  from  the  air. 
He  added  some  drops  of  metallic  quicksilver  or  iodine  solution 
to  it  in  order  to  make  it  keep.154  He  found,  furthermore,155 
that  the  nucleus  with  nitrate  of  silver  was  colored  almost  black 
under  the  influence  of  light,  and  that  when  it  was  laid  first  in  a 
dilute  solution  of  ferrocyanide  of  potassium,  then  carefully 
washed  out  and  treated  with  a  dilute  solution  of  ferric  chloride  it 

IBS  Behrens  in  Flora,  1879,  p.  444,  ff. 
is8  Hanstein,  I.  c.,  Taf,  XI,  Figs.  17,  23. 
»4  Hartig  in  Bot.  Zeitg.,  1854,  p.  877. 
"6 Hartig,  I.  c.,  p.  878. 


398  THE  MICROSCOPE  IN  BOTANY. 

was  colored  a  deep  blue.  But  if  we  apply  Berlin  blue  direct, 
the  nucleus  becomes  not  blue  but  a  pale,  smutty,  reddish  color. 
If  in  addition  to  this  we  note  that  it  was  known  that  the  nucleus 
became  distinct  in  acetic  acid,  and  thus  was  first  made  visible, 
but  that  by  the  use  of  concentrated  acid  it  was  made  to  swell, 
we  have  exhausted  pretty  nearly  all  the  histological  nucleus- 
reagents  of  the  older  authors. 

In  later  times  we  aim  at  two  things  in  the  use  of  these  rea- 
gents:  (1)  fixing  the  nucleus;  (2)  rendering  it  visible  by 
staining.  We  will  particularly  consider  both. 

A.    FIXING  THE  STRUCTURE  OF  THE  NUCLEUS. 

The  fixing  (or  "setting")  is  done  by  very  dilute  organic  or 
inorganic  acids.  In  almost  all  cases  the  fixing  takes  place  very 
quickly  so  that  the  preparation  fteed  remain  but  a  short  time  in 
the  reagent.  Acetic  acid  works  very  well  and  a  solution  of  not 
higher  than  one  per  cent  should  be  used.  It  then  produces  no 
shrinking  of  the  nucleus  but  its  stringy  framework  comes  out  very 
distinctly.  By  the  use  of  stronger  acid  the  swelling  produced 
makes  it  again  very  soon  quite  indistinct.  In  place  of  the  acetic 
one  may  use  formic  acid  (Retzius).  For  other  cases  chromic 
acid  gives  excellent  service  in  a  |-  to  £  per  cent  solution,  some- 
times even  as  strong  as  a  one  per  cent  solution.156  Picric  acid 
may  also  be  employed  in  different  degrees  of  dilution.  Nitric 
or  picro-sulphuric  acids  are  less  worthy  of  commendation  since 
they  occasion  a  considerable  shrinking,  the  preparation  becoming 
much  less  beautiful  than  in  chromic  acid.  On  the  other  hand 
osrnic  acid  is  a  very  excellent  fixing  medium  used  in  a  one  per 
cent  solution.157  It  makes  the  structure  of  the  nucleus  very 
distinct  but  in  many  cases  indeed  causes  it  to  swell.  Strasbur- 
ger  used  it,  for  example,  in  following  out  the  division  of  the 
nucleus  in  the  mother  cell  of  the  pollen.  He  emptied  the 
pollen  sack  into  a  3  per  cent  solution  of  sugar  and  added  a 
drop  of  1  per  cent  solution  of  osmic  acid,  when  all  the  relations 

«8  Strasburger,  Zellbildung  u.  Zelltheilung,  pp.  172, 173,  etc. 
167  Strasburger,  I.  c.,  p.  39  und  anderwarta, 


STAINING  THE  NUCLEUS.  399 

came  out  sharply  after  some  minutes.158  Fleisch159  has  proposed 
for  a  like  purpose  a  mixture  of  chromic  and  osmic  acid,  which 
according  to  Fleming  fixes  it  well  enough  but  the  structure 
appears  pale  and  is  stained  with  difficulty.  But  this  objection 
according  to  Fleming160  is  obviated  if  one  adds  a  little  acetic 
acid  to  the  mixture.  Then  a  very  beautiful  staining  with  hem- 
atoxylin,  picro-carmine,  and  gentiana  is  obtained.  Fleming's 
mixture  consists  of  chromic  acid,  0.25  per  cent,  osmic  acid  0.1 
per  cent  and  acetic  acid  0.1  per  cent  in  distilled  water. 

[Absolute  alcohol  fixes  the  protoplasm  without  contracting 
it.  The  section  or  the  whole  organ  may  be  plunged  into  it. 
Strasburger  by  putting  Spirogyra  orthospira  in  absolute  alcohol 
at  different  hours  of  the  night  succeeded  in  fixing  the  division 
of  the  nuclei  of  this  alga  in  their  various  stages  of  development 
so  they  could  be  studied  the  next  day  very  easily.  The  same 
observer  also  retarded  the  development  of  the  nuclei  till  morn- 
ing by  placing  the  plant  in  a  room  without  heat  in  November 
and  so  could  watch  the  development  by  daylight  and  fix  them 
at  the  most  suitable  moment.  A.  B.  H.j 


B.    STAINING  THE  NUCLEUS. 

By  the  use  of  these  means  the  structure  of  the  nucleus  is 
fixed,  that  is,  becomes  distinct.  We  now  proceed  to  stain  it. 
This  may  be  done  with  the  aniline  dyes  (p.  299,^*.),  haematoxy- 
liu  (p.  304),  cochineal  extract  (p.  305),  or  carmine  solutions 
(which  see).  Those  most  worthy  of  commendation  are  the  fol- 
lowing. (The  methods  of  preparing  these  staining  media  have 
already  been  given.) 

1 .  Staining  with  Borax-carmine  (Strasburger)  .16]  The  sec- 
tion commonly  needs  to  lie  in  the  mixture,  described  on  p.  307, 
but  a  short  time.  Concerning  the  examination  and  preservation 
of  the  specimen  see  that  page. 

158  Strasburgev,  1.  c.,  p.  21.  For  a  like  purpose  Hartig,  earlier,  treated  pollen  grains  with 
carmine  glycerine  twelve  to  twenty-four  hours  (Dot.  Unters.  herausgeg.  v.  Karsten,  1866, 
HeitS,  p.  249). 

«9  Fleisch  in  Arch.,  f.  mikrosk.  Anat.,  Bd.  XVI,  p.  300. 

160  Fleming,  Zellsubstancc,  Kern  und  Zelltheiluug,  Lpz.,  1882,  p.  381. 

161  Strasburger,  1.  c  ,  p.  9. 


400  THE  MICROSCOPE  IN  BOTANY. 

2.  Staining   with  Beale's  carmine  (Strasburger) .       Com- 
mended  for   filamentous   algoe.     For  particular  directions  see 
p.  307. 

3.  Staining   with  Acetic  acid  carmine    (Fleming).      This 
fluid  is  suitable  only  for  fresh  sections,  which  sometimes  be- 
come very  beautiful  in  it. 

4.  Staining  with  Picro-carmine  (Fleming,  Treub).     Alike 
commendable  for  animal  and  vegetable  tissue.     The  section  needs 
to  lie  in  the  fluid  but  a  very  short  time.     Mount  in  glycerine. 

5.  Staining  with  Hcematoxylin  (Frey,  Strasburger,  Flem- 
ing).    Those  sections  which  have  been  fixed  in  osmic  acid  and 
have  been  freshly  washed  are  especially  to  be  commended  for 
this  staining  medium,  as  they  then  take  up  the  coloring  matter 
well.     If  they  have  lain  in  alcohol  for  a  long  time  they  color 
badly.     Staining  may  be  done  in  either  a  strong  or  dilute  solu- 
tion ;  in  the  latter  case  it  will  require  from  twenty-four  to  forty- 
eight  hours.    If  the  section  becomes  over-colored,  alum  water  or 
dilute  muriatic  acid  will  clear  it  up ;  in  the  use  of  the  latter  the 
nucleus  will  be  slightly  swollen.     Make  use  of  the  hsematoxylin 
solution  given  by  Frey  or  one  of  the  new  things  recommended 
by  Grenadier.162 

6.  Staining  with  Picro-hoematoxylin.1®     This  as  I  can  tes- 
tify from   some    experiments   of  my  own  is    a    very  superior 
method  and  is  described  by  Schmitz  as  follows.     The  section  of 
the  fresh  plant  is  put  in  a  concentrated  solution  of  picric  acid  and 
remains  for  a  shorter  or  longer  time,  even  over  night  if  neces- 
sary.     In  this  picric  acid  solution  the  protoplasm  immediately 
hardens.     By  longer  continuance   in  the  solution  the  plasmic 
part  of  the  cells  contracts  a  very  little,  but  that  is  in  many  cases 
an   advantage  to  the  investigation,  for  by  this  means  the  cell 
membrane  becomes    much    more    transparent   to  the  coloring 
matter  of  the  plasma  without  itself  becoming  stained.     As  a 
coloring  matter  for  the  plasmic  body  I  now  almost  always  use 
hsematoxylin    in   an  aqueous   solution  without  the  addition  of 

162  Prepare  a  saturated  solution  of  crystallized  haematoxyliu  in  absolute  alcohol  and  a 
like  one  of  ammoniacal  alum  in  water.  Mix  4  cc.  of  the  former  with  150  cc.  of  the 'lat- 
ter. Let  it  stand  for  a  week  in  the  light,  filter  and  add  25  cc.  of  glycerine  and  25  cc.  of 
mythel  alcohol.  After  all  the  free  precipitate  has  settled  the  reagent  is  ready  for  use. 

16»  Schmitz,  in  Sitzungsb.  der  niederrhein.  Gescllsch.  zu  Bonn,  1880,  p.  160. 


STAINING  THE  NUCLEUS.  401 

alum.  I  lay  the  object  in  water,  it  having  been  freed  from 
every  trace  of  picric  acid  by  repeated  and  careful  washings,  and 
add  a  small  quantity  of  hsem-itoxylin  which  has  absorbed  am- 
monia from  the  air  and  so  is  partly  changed  into  hiBinatein-am- 
monia.  The  coloring  matter  dissolves  rapidly  in  pure  water 
with  a  red  color  and  gives  a  solution  which  gradually  darkens 
and  after  some  time  decomposes.  After  remaining  for  some 
time  (from  one  to  several  hours)  in  the  solution,  whose  degree 
of  concentration  must  be  chosen,  according  to  the  special  pur- 
pose in  view,  the  object  should  be  taken  out  and  washed  in 
water  till  the  wash  water  remains  quite  colorless.  Then  the 
object  will,  according  to  the  quantity  of  the  coloring  matter 
used  and  the  length  of  time  given  to  its  effect  (this  must  be 
tested  for  each  case),  be  colored  blue,  in  more  or  less  intense 
shade,  either  the  eliminate  bodies  of  the  nucleus  alone,  or  these 
and  the  rest  of  the  substance  of  the  nucleus,  as  also  the  thicker 
plasmic  bodies,  as  for  example  the  crystalloids,  or  the  whole  of 
the  plasmic  elements  of  the  cell ;  but  the  whole  cell  membrane, 
starch  grains,  oil  drops  and  crystals  remain  almost  colorless. 
The  color  is  best  preserved  when  the  preparation  is  mounted 
in  glycerine,  but  one  must  be  absolutely  sure  that  not  a  trace 
of  free  acid  remains  in  the  specimen.  The  least  particle  of 
acid  will  infallibly  destroy  the  color  in  time  and  very  provok- 
ingly  render  the  most  excellent  specimens  useless. 

7.  'Staining  with  Methyl  green.      (Strasburger,  Fromann, 
Fleming.)     Strasburger164  uses  a  one  per  cent  solution  of  acetic 
acid  which  he  dilutes  with  methyl  green  (commended  by  Mey- 
zel).     In  order  to  see  the  form  of  the  nucleus  of  the  pollen 
mother  cell,  he  puts  a  young  anther  in  the  acetic  methyl  green 
solution  and  breaks  it  open   by  pressure.     The  outcoming  con- 
tents are  immediately  fixed  and  the  figure  of  the  nucleus  be- 
comes at  once  beautifully  stained  by  the  methyl  green.      Alas  ! 
that  a  preparation  so  made  should  not  keep. 

8.  Staining  with  other  Aniline  coloring  substances.     Among 
the  many  coloring  substances  here  commended  may  be  specially 
mentioned  Saffranin,  Dahlia,   Gentiana  violet,  the  latter  with 

164  Strasburger,  1.  c.,  p.  141. 


402  THE  MICROSCOPE  IN  BOTANY. 

acetic  acid,  as  affording  very  beautiful  colors.  Aniline  prepara- 
tions are  best  preserved  in  dammar  varnish  or  Canada  balsam, 
but  they  shrink  if  they  are  for  this  purpose  previously  put  in 
oil  of  cloves.  Fleming165,  therefore,  recommends  mounting 
them  m  resinous  turpentine  oil  after  previously  passing  through 
dilute  and  then  absolute  alcohol  with  which  one  may  gradually 
mix  the  turpentine  oil. 

\_Pfitzer's  Reagent  for  simultaneous  Staining  and  Hardening. 
E.  Ptitzer  has  reported  a  fluid  which  both  hardens  and  stains 
vegetable  protoplasm.-  It  consists  of  the  coloring  matter, 
nurrosin,  dissolved  with  picric  acid  in  water  or  alcohol.] 

\_(a)  To  a  concentrated  solution  of  picric  acid  is  added  a 
small  quantity  of  an  aqueous  solution  of  nigrosin.  If  the 
.object  to  be  studied  contains  much  water  some  crystals  of  the 
acid  should  be  added  to  maintain  the  strength  of  the  liquid.] 

[The  deep  olive-green  fluid  kills  with  great  rapidity.  After 
some  hours'  immersion  of  the  object  which  is  to  be  examined  it 
may  be  transferred  to  alcohol,  especially  if  it  be  desired  to 
dissolve  out  the  chloiophyll,  or  if  the  object  has  to  be  kept  some 
time.  By  this  means  the  denser  masses  of  protoplasm  are 
stained  pale  violet,  the  chroinatophores  darker,  while  the 
pyrenoid,  nucleoli  and  other  colored  parts  of  the  nucleus  come 
.out  deeply  stained ;  thin  films  of  protoplasm  and  ordinary 
cellulose  membranes  are  scarcely,  if  at  all,  stained;  starch 
.grains  not  at  all.  By  washing  the  objects  in  water  after  stain- 
ing instead  of  in  spirits,  a  gray-blue  color  is  obtained ;  trans- 
ference to  strong  glycerine  makes  the  color  purer.  The  color 
comes  out  best  however  alter  washing  in  alcohol,  treating  with 
oil  of  cloves  and  mounting  in  one  of  the  resins,  dammar  or 
Canada  balsam] . 

[To  avoid  contraction  the  clove  oil  may  be  diluted  with  alcohol 
and  allowed  to  concentrate  upon  the  object  by  evaporation  of 
the  alcohol.  The  watery  solution  is  especially  adapted  for 
rapidly  killing  and  staining  objects  already  under  the  micro- 
scope.] 

[(&)  Nigrosin  and  picric  acid  may  also  be  used  in  solution  in 
alcohol.  The  solid  acid  and  nigrosin  are  left  for  some  time  in 

"*  Fleming,  L  c,,  p.  384. 


IMPORTANT  REACTIONS  FOR  FLUID  CONTENTS  OF  CELLS.  403 


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404  THE  MICROSCOPE  IN  BOTANY. 

absolute  alcohol ;  by  this  solution  the  chromatophores  and  pyr- 
enoids  are  less  deeply  stained ;  the  colored  contents  of  the 
nucleus  very  deeply  so.] 

[Quoted  from  Jour.  Roy.  Microscop.  Soc.,  Vol.  Ill,  No.  Ill, 
pp.  445-6.     A.  B.  H.]. 


Under  the  headings  III,  IV,  Y,  VI,  VII,  VIII  and  IX  we 

have  treated  those  frequently-occurring  substances,  the  contents 
of  cells,  which  appear  to  be  colorless  fluids  or  very  like  such. 
As  with  the  solid  framework  of  the  cell  (see  p.  356)  so  with 
these  we  also  tabulate  the  principal  reagents  used  in  their 
identification  (p.  403). 

X.  CHLOROPHYLL  (LEAF-GREEN). 

Literature,  v.  Mohl,  Vermischte  Sch.  Tub.,  1845,  p.  352, 
ff.  (Unters.  iiber  d.  Anat.  Verhalten  d.  Chlorophylls).— v. 
Mohl,  Ueber  d.  Bau  d.  Chloroph.  (Bot.  Zeitg.,  1855,  p.  89, 
ff. ). — Bohm,  Beitr.  z.  naheren  Kcnntn.  d.  Chi.  (Sitzungsber. 
d.  K.  Acad.  d.  Wiss.  Wien,  Bd.  XXII,  1857,  pp.  479-512). 
Gris,  Recherch.  microsc.  sur  la  chl.  (Ann.  des  sc.  nat.,  4e  ser., 
t.  VII,  1857,  pp.  179-219).— Hartig,  Entwicklungsgeschichte 
d.  Pflankeims.,  Leipzig,  1858,  pp.  79-82. — Morren,  Dtss.  sur 
les  feuilles  vertes  et  colorees,  1858. — Sachs,  Ueber  d.  Ergeb- 
nisse  einigeneu.  Unters.  iiber  d.  Chl.  (Flora,  1862,  p.  129,^".). 
— Sachs,  Ueber  d.  Einfl.  d.  Lichtes  auf  d.  Bild.  d.  Arnylums 
in  d.  Chlkornern  (Bot.  Zeitg.,  1862,  p.  364,  /.).— Sachs, 
Beitr.  z.  Physiol.  d.  Chl.  (Flora,  1863,  p.  193,  /.).— Sachs, 
Ueber  d.  Auflos.  ti.  Wiederbild.  d.  Amylums  in  d.  Chlk.  (Bot. 
Zeitg.,  1864,  p.  289,  ff.). —Sachs,  Handbuch  d.  Experimental- 
phys.  d.  Pfl.,  Lpz.,  1865,  pp.  309,/".,  313,  ff.  —  (Nageli  u. 
Schwendener,  Mikros.,  Lpz.,  1867,  p.  496). — Micheli, 
Quelq.  obser.  sur  la  matiere  col.  de  la  chl.  (Arch.  d.  sc.  d.  la 
bibliothe.  univ.  de  Geneve,  Mai,  1867). — Dippel,  Mikrosk., 
Bd.  II,  p.  32,  ff.— Kraus,  G.,  Einige  Beob.  ttber  d.  Einfl.  d. 
Licht.  u.  d.  Warme  auf  die  Entsteh.  d.  Starkeerzeugung  im 
Chlorophyll  (Pringsheim's  Jahrb.,  Bd.  VII,  p.  511,  ff.).— 


CHLOROPHYLL  (LEAF-GREEN.)  405 

Wiesner,  Chi.  in  Xeottia  Nidus-avis  betr.  (Bot.  Zeitg.,  1871, 
p.  619).— Kraus,  G.,  Z.  Kcnntu.  d.  Chlorophyllfarbst.  u. 
ihrer  Verwandten.,  Sttittg.,  1872. — Wiesner,  Unters.  iiber  d. 
FarbstofFe  einiger  fur  chlorophyllfroi  gekaltenen  Phanerog. 
(Pringsheim's  Jahrb.,  Bd.  VIII,  1872,  p.  575,  ff.).— -Kraus, 
G.,  Einige  Bemerk.  iiber  d.  Erscheiimngd.  Soinmerdiirre  unser- 
er in  Baura-  und  Strauchblatter  (Bot.  Zeitg.,  1873,  p.  401,  ff.). 
— Briosi,Uebernormale  Bild.  v.  fettart.  Subst.  im  Chlrophyll. 
(id.,  p.  529,  ff.).—  Drude,  D.  Biol.  v.  Neottia  Nidus-avis  u. 
Monotropa  hyp.  Gottingen,  1873. — Kraus,  G.,  Ueber  d.  Ur- 
sache  d.  Farbung  d.  Epidermis  vegetat.  Organe  d.  Pfl.  (Flora, 

1873,  p.    316,  /.).— Treub,  Z.  Chlorophyllfrage   (id.,   1874, 
p.  55,   /.) — Wiesner,    Bemerk,    iiber  d.    angelb.    Bestandth. 
des  Chi.  (id.,  p.  278,^.). — Prill ieux,  sur  la  color,  et  leverdiss. 
du   Neottia   Nidus-avis    (Ann.  des  sc.  nat.,  5eser.,   t.  XIX, 

1874,  p.  109,  ff.).— Batalin,   Ueber  d.  Zerst.  Chloroph.  in  d. 
leb.    Organen    (Bot.    Zeitg.,    1874,    p.    433,  ff.) .—Wiesner, 
Vorl.  Mitth.   iiber  d.  Einfl.  d..Lichtes  auf  Entsteh.   u.  Zerstor. 
d.  Chi.  (id.,  p.  11 6,  ff.).— Sachs,  Lehrb.,  p.  47,  ff.— Wies- 
uer,  Welche  Strahlen  des  Lichtes  zerlegen  bei  Sauerstoffzutritt 
d.    Chloroph.?  (PoggendorflTs  Aiinalen,   Bd.    CLIl,   1874,  p. 
496,  ft.). — Pringsheim,  Ueber  d.  Absorptionsspeetra  d.  Chloro- 
phyllfarbst.  (Monatsber.  K.  Acad.  Berlin,  1874,  pp.  628-659). 
— Pringsheim,   Ueber    d.      natiirl.    Chlorophyllmodificationen, 
etc.     (id.,    1875,    pp.    745-759). — Askenasy,   Ueber  d.    Zer- 
stor. d.   Chi.   leb.  Pfl.  clurch  d.  Licht.  (Bot.   Zeitg.,  1875,  p. 
457, /*.).—  Haberlandt,  Ueber  d.  Einfl.  d.  Frostes  auf  d.  Chlk. 
(Oesterr.  Bot.  Zcitschr.,  187(5,  p.  249,^.).— Haberlandt,  Ueber 
d.  Entsteh.   d.   Chi.  in  d.   Keimbl.   v.  Phaseolus  (Bot.  Zeitg., 
1877,  p.  3(U,jf.).--Sachsse,  Chem.  u.  Phys.   d.   Farbstotto, 
Kohlehyclrate,  etc.,  Lpz.,  1877. — Dippel,  Einige  Bemerk.  iiber 
d.  Gemength.  d.   Chlorophylls,  etc.  (Flora,   1878.  p.    11,  ff.). 
Hoppe-Seyler,  Ueber  d.  Chloroph.  d.  Pfl.  (Bot.  Zeitg..  1879,  p. 
815,^.). — Pringsheim,  Ueber  d.  Lichtwirk.  u.   Chlorophyll- 
function  in  d.  Pfl.  (Bot.   Zeitg.,  1879,  p.  789,  ff.).—pYi^. 
sheim,  Ueber  Lichtwirk.  u.  Chlfunction  in  d.  Pfl.  (Monatsber. 
K.  Acad.  Berlin,  1879,  Juli,   17  pp.)- — Pringsheim,  Ueber  d. 
Hypochlorin  u.  d.  Bedingungen  s.  Entst.  in  d.  Pfl.  (id,  1879, 


406  THE  MICROSCOPE  IN  BOTANY. 

Nov.,  21  pp.)- — Flahault,  Stir  la  presence  de  la  mat.  verte  dans 
les  org.   actuellem.   soustraits  a  1'infl.  de  la  him.  (Bull,  de  la 
Soc.  bot.  de  France,  t.  XXYI,  p.  249,  /.).— Pringsheim,  Z. 
Kritik  d.  bisherig.  Grundlagen  d.   Assimilationstheorie  d.  Pfl. 
(Monatsber.  d.  K.  Acad.  Berlin,  1881,  pp.  117-135).—  Prings- 
heim,    Ueber   d.    primare   Wirk.    d.    Lichtes   auf    d.    Veget. 
(id.,   pp.  504-535). — Pringsheim,  Ueber  Lichtwirk.  u.  Chl- 
funct.  in  d.  Pfl.  (Pringsheim's  Jahrb.,  Bd.  XII,  1881,  p.  288, 
ff.). — Hansen,   Gesch.  d.   Assimilationstheorie  u.  Chlorophyll- 
function.,  Lpz.,  1882  (auch  Arb.  Bot.  Inst.  Wiirzbg.,  Bd.  II, 
Heft.  4). — Tschirch,   Unters.  liber  d.  Chloroph.  (Botan.  Cen- 
tral bl.,  Bd.  XI,  1882,  p.   107, /".).—  Wiesner,  Bemerk.  liber 
d.  Natur  d.  Hypochlorins   (id.,  Bd.  X,  1882,  p.  260,  /*.).- 
Piingsheim,  Ueber  Chlorophyllfunct.   u.   Lichtwirk.  in  d.  Pfl. 
(Pringsheim's  Jahrb.,  Bd.  XIII,  Hett  3;  116  pp.).— Schim- 
per,    Ueber   d.    Gestalten    d.  Starkebildner    und    Farbkorper 
(Botan.  Centralb.,  Bd.  XII,  1882,  p.  1 75,  ff.).— Meyer,  Ueber 
Chlorophyllk.,  Starkbildner  u.  Farbk.  (id.,  p.  314,  ff.). 

tipectroscopic   behavior  of  Chlorophyll.      Brewster,    on    the 

color  of  natural  bodies  (Transact.  Roy.   Soc.  of  Edinburgh,  t. 

XII,  1834,  p.  538,  ff.)     Angstrom,  Ueber  d.  grtine  Farbe  d. 

Pfl.   (Poggendort's  Annalen,  Bd.    XCIII,  1854,  p.  475,  jf.), 

also  (Ofversigt  af.  K.  Ventesk.  Acad.  Forhantll,  1853,  p.  246, 

2f.). — Stockes,  Ueber  d.   Verand.    d.    Brechbark.    d.    Lichtes 

(Poggendorff's   Ann.,  Erganzungsbd.  IV,  1854,  pp.  217-228). 

— Harting,  Ueber  d.  Absorptionsvermogen  d.  reinen  und  un- 

reinen    Chlorophyll   fur   die    Strahlen    der    Sonne     (id.,    Bd. 

XCVI,  1855,  p.  543,/l). — Askenasy,  Beitr.  z.  Kenntn.  d.  Chi. 

u.  einigerdass.  begleit.  Farbst.  (Bot.  Zeitg.,  1867,  p.  225,/.). 

— Sorby.     On  a  definite  method  of  qualit.  analysis  of  aniin.  and 

vegetable  coloring  matters  by  means  of  the  spectro-microscope 

(Proceed,  of  the  Roy.  Soc.  of  Loud.  Vol.  XV,  1867,  p.  433- 

436). — Hagenbacb,   Unter?.    liber    d.    opt.   Eigenschaften    des 

Blattgruns  (Poggendorff's  Ann.  Bd.  CXLI,  1870,  p.  245-275). 

— Gerland  et  Rauwenhoff,  Rcch.  sur  la  Chlorophyll  et  quelques- 

uns  de  ses  derives  (Arch,  neerland.,  t.  *VI,  1871,  p.  97,  ff.).— 

Sorby,  Various  tints  of  autumnal  foliage  (Quarterly  Journ.  of 

Science,   No.   XXIV,    Jan.    1871,    p.    64-77). — Kraus,    Zur 


CHLOROPHYLL  (LEAF-GREEN).  407 

Kemitu.  tier  Chlorophyllfarbstoffe,  Spectralunalyt.  "[Inters. 
Stuttg.,  1872. — Sorby,  On  comparative  vegetable  Chromatol- 
ogy  (Proceed.  Royal.  Soc.  of  Loud.,  Vol.  XXI,  1873,  p.  442- 
483). — Pringsheim,  Uebcr  d.  Absorptionsspectra  d.  Chlfarb- 
stoffe  (Mouatsber.  d.  K.  Acad.  d.  Wiss.,  Berlin,  1874,  p. 
628-G59) . — Pringsheim,  Ueber  natiirl.  Chlorophyllmodifica- 
tionen,  etc.  (id.,  1875,  p.  745-759).— Pringsheim,  Ueber 
Lichtwirk.  u.  Chlomphyllfunction  in  d.  Pfl.  (Pringsheim's 
Jahrb.,  Bd.  XII,  1880, p.  408,  jf.).166  [C.  Timiriazeff,  Coinptcs 
Kendus  XCVI,  1883,  pp.  375-6.] 

Chlorophyll  or  leaf  green  very  seldom  occurs  in  cells  in  a  pure 
state,  that  is,  as  a  dissolved  green  pigment  (Hildebraud,  Weiss, 
Trecul ;  the  cases  are,  however,  still  doubtful  because  they  have 
not  been  exactly  investigated),  but  mostly  in  combination  with 
proteid  substances.  The  latter  commonly  form  grains,  more 
rarely  spiral  bands  (Spirogyra),  or  star-shaped  forms  (Zyg- 
nema).  They  represent  the  colorless  fundamental  substanc3 
which  is  covered  or  penetrated  by  the  green  coloring  matter. 
On  account  of  their  prevailing  granular  form  we  commonly  speak 
of  chlorophyll  grains.  But  the  chlorophyll  can  easily  be  separa- 
ted from  the  fundamental  substance  (by  alcohol,  benzole,  etc., 
in  which  it  dissolves)  the  colorless  mass  being  left  without 
having  its  form  perceptibly  changed.  It  consists  as  already 
mentioned  in  great  part  of  albuminous  substances  but  contains 
also  small  quantities  of  fats,  oils,  tannic  acid  and  sugar.  As 
is  well  known  the  chlorophyll  grains  arc  the  seat  of  the  process 
of  assimilation.  In  them  starch  is  formed,  in  a  manner  still 
unknown,  from  the  elements  of  carbonic  acid  and  water,  as  the 
first  visible  product  of  assimilation  (in  many  cases  fat-like  sub- 
stances in  place  of  this,  Sachs,  Briosi) .  Till  very  recently  starch 
has  been  considered  the  first  visible  product  of  assimilation, 
but  according  to  the  more  recent  investigations  of  Pringsheim 
the  first  product  is  a  body  containing  oil,  called  Hypochlorine, 
which  may  be  separated  by  muriatic  acid  and  soon  assumes 
crystal-like  strata.  It  penetrates  the  whole  porous  proteid 
framework  of  the  grain.  Sachs,  Hansen,  Tschirch  deny  the 

"6  See  also  note  144  on  p.  334.    A  perfect  list  of  the  chemical  literature  concerning  chlo- 
rophyll may  be  found  in  lliiseinann,  p.  24,  ff. 


408  THE  MICROSCOPE  IN  BOTANY. 

existence  of  hypochlorine,  asserting  it  to  be  the  product  of 
the  effect  of  the  acid  upon  the  chlorophyll.  The  investigations 
of  this  subject  are  not  yet  concluded.  That  starch  may  easily 
be  made  visible  in  chlorophyll  grains  we  have  already  shown 
(p.  364). 

In  the  microscopical  investigations  of  chlorophyll  grains  they 
should  be  so  treated  as  to  study  either  the  colorless  ground- 
substance,  or  the  coloring  matter  itself.  We  will  consider  both 
in  their  turn. 

1.     THE  FUNDAMENTAL  SUBSTANCE  OF  CHLOROPHYLL 

GRAINS- 

The  fundamental  substance  imy  be  investigated  in  the  gran- 
ules which  are  permeated  with  coloring  matter,  also — and 
indeed  much  better — in  those  which  have  been  freed  from  the  , 
chlorophyll.  For  the  latter  purpose  the  portion  of  the  plant  to 
be  investigated  or  a  section  of  it  should  be  laid  in  at  least  90 
per  cent  alcohol  or  in  ether  which  will  dissolve  out  the  coloring 
matter  and  bleach  the  object.  It  is  not  recommended  to  boil 
the  specimen  in  water  as  in  preparing  a  chlorophyll  solution 
since  this  coagulates  the  fundamental  mass.  The  bleached 
grains  remain  behind  quite  unchanged  in  the  cells.  Those  chlo- 
rophyll grains  which  contain  no  starch  are  best  adapted  to  this 
investigation  (for  example,  those  of  Allium  cepa  Sachs),  other- 
wise one  would  naturally  get  the  characteristic  reaction  of  starch. 
Many  reactions,  however,  are  uninjured  by  the  presence  of 
starch. 

Of  the  microscopical  reactions  the  following  may  be  referred 
to.167  The  bleached  grains  take  a  brick  red  color  from  acetic 
acid  cochineal  extract  (Maschke)  :  alcoholic  iodine  solution 
colors  them  yellow  or  dark  brown  with  contraction.  Put  a 
section  containing  the  bleached  grains  in  a  concentrated  solution 
of  copper  sulphate  for  about  half  an  hour,  wash  and  transfer  to 
a  strong  solution  of  potash  and  the  grains  become  a  distinct 
violet.  Similar  sections  somewhat  warmed  in  nitric  acid, 
washed  out  with  water,  and  treated  with  potash  solution,  the 

167  Mostly  according  to  Sachs,  Flora,  1863,  p.  195,  ff. 


THE  FUNDAMENTAL  SUBSTANCE  OF  CHLOROPHYLL.      409 

chlorophyll  grains  will  either  retain  their  form  or  be  changed 
into  a  formless  mass.  In  the  former  case  each  chlorophyll  grain 
becomes  a  distinct  orange  yellow ;  in  the  latter  case  the  cell 
is  filled  with  an  orange  yellow,  amorphous  mass.  Green  leaves 
laid  in  a  concentrated  solution  of  potash  for  about  an  hour,  the 
chlorophyll  grains  remain  green  and  unchanged ;  wash  with 
water  and  the  cells  will  contain  a  homogeneous  mucilage;  neu- 
tralize with  acetic  acid  and  add  alcoholic  iodine  and  the  cells 
will  appear  to  be  filled  with  a  fine  granular  brownish  mass. 
If  the  leaves  lie  for  several  days  in  the  potash  the  chlorophyll 
will  run  together  into  a  homogeneous  layer.  Bleached  chloro- 
phyll grains  behave  under  this  treatment  quite  the  same  only 
that  they  are  more  resistant  to  the  action  of  the  strong  al- 
kali than  the  green.  Ammonia  leaves  the  form  in  fresh  gran- 
ules quite  distinct,  the  substance  only  becoming  somewhat  filled 
with  spaces  and  vacuoles.  After  washing  they  are  still  almost 
exactly  the  same  green  ;  neutralize  with  acetic  acid  and  add 
alcoholic  iodine  and  the  granules  appear  sharply  defined,  some- 
what contracted  with  vacuoles,  brown.  In  other  cases  they  are 
less  resistant  toward  ammonia.  Phosphoric  acid  makes  fresh  chlo- 
rophyll grains  yellow,  but  does  not  change  the  bleached  grains 
at  all.  The  gi-een  as  well  as  the  bleached  grains  are  much  more 
resistant  toward  sulphuric  acid  than  is  either  protoplasm  or  the 
cell  nucleus ;  the  green  ones  become  either  verdigris  or  blue 
green.  Cold  acetic  acid  colors  green  chlorophyll  grains  clear 
yellow  but  leaves  their  form  unchanged  ;  by  boiling  in  it  they  be- 
come knotty. 

We  infer  from  all  these  reactions  that  the  fundamental  sub- 
stance of  chlorophyll  grains  belongs  to  nitrogenous  matter. 
It  is  a  "protoplasmic  form"  (Sachs). 

According  to  Pringsheim,168  treating  chlorophyll  grains  with 
dilute  muriatic  acid  (the  best  is  one  part  acid  to  four  parts 
water)  for  a  long  tinie  they  become  a  yellow-green,  gold  yel- 
low or  brownish.  After  a  longer  time  (several  hours)  their 
dark,  reddish-brown  or  rust-colored  periphera  separates  from 
the  rest  of  the  substance  of  the  sharply  defined  mass  of  the 
chlorophyll  grain.  This  becomes  afterwards  distinctly  angular, 

,  las  Pringsheim's  Jahrb.,  Bd.  XII,  p.  294. 


410  THE  MICROSCOPE  IN  BOTANY. 

pointed  and  forms  a  more  or  less  extended  scale  or  nest  of  in- 
distinct crystal-like  forms  which  throw  out  oblique  and  pointed 
projections.  They  are  Hypochlorin  and  correspond  to  a  mix- 
ture of  oil  and  resin  substances.  They  are  insoluble  in  water 
and  dilute  acid ;  on  the  other  hand  they  are  soluble  in  ether, 
benzole  and  sulphuretted  carbon,  and  vaporize  at  about  50°. 

2.    CHLOROPHYLL  COLORING  MATTER. 

'Notwithstanding  the  many  investigations  into  the  nature  of 
the  coloring  matter  of  chlorophyll  it  is  not  yet  satisfactorily 
known.  By  the  latest  investigations  it  appears  to  be  established 
that  crude  chlorophyll  (Rohchlorophyll,  Wiesner)  consists  of 
at  least  a  yellow  and  a  green  coloring  matter  (chlorophyll  in 
the  strict  sense).  The  chemical  composition  of  the  green  col- 
oring matter  is,  according  to  Gautier,  C  =  73.97,  H  =  9.80,  O 
=  10.33,  N  =  4.15,  incombustible  elements  =  1.75.  With 
this  essentially  agrees  the  analysis  of  Rogalski,169  while  those 
of  others  are  very  different.  Essential  chlorophyll  is  accord- 
ing to  Gautier170  a  crystal  I  izable  body  ;  he  obtained  clinorhombic 
crystals  about  £  cm.  long  of  soft  consistency  and  intense  green 
color  which  in  the  light  became  yellow-brown,  yellowish  or 
brownish  and  afterwards  quite  colorless. 

In  the  study  of  chlorophyll  coloring  matter  we  must  investi- 
gate its  chemical  reaction,  as  well  as  its  optical  (spcctroscopic) 
behavior. 

A.     Behavior  toward  Reagents. 

With  the  exception  of  a  yellow  or  greenish  decomposition 
product171  which  crude  chlorophyll  often  forms  in  water  and  is 
soluble  in  that,  it  is  insoluble  in  cold  or  boiling  water  as  well 
as  in  dilute  acids  or  alkalies.  On  the  other  hand  it  is  soluble 
in  alcohol,  sulphuretted  carbon,  ether,  benzole  (Kraus),  in 
many  oils  and  in  turpentine  (Wiesner).  In  order  to  prepare 
an  alcoholic  solution  of  crude  chlorophyll  the  part  of  the  plant 

169  Cf.  Husemann,  1.  c.,  p.  251. 

170  Gautier  in  Comptes  Kendus,  LXXXIX,  p.  861,  ff. 

171  Pringsheim  inHouatsber.  d.  K.  Acad.  d.  Wiss.  Berlin,  1875,  p.  748. 


CHLOROPHYLL  REACTIONS.  411 

to  be  used,  preferably  leaves,  should  be  (according  to  Kraus)172 
put  in  hot  water  and  boiled  once  or  twice,  the  water  poured 
off  and  boiling  alcohol  of  95  per  cent  (sp.  w.  0.816)  poured 
on.  If  alcohol  of  83  per  cent  be  used  cold,  the  parts  of  the 
plant  containing  oils,  wax,  etc.,  will  not  enter  into  the  solution 
(Gautier).  The  alcoholic  crude  chlorophyll  extract  should 
be  fresh  when  used  in  investigations,  although  it  is  much 
less  decomposable  if  the  leaves  have  been  previously  boiled ; 
apparently  this  manipulation  removes  the  salts  and  other  im- 
purities from  the  leaves  (Stockes,  Kraus).  The  chlorophyll 
solution  thus  prepared  is  of  a  beautiful  green  color  and  has  a 
dark  red  luster.  It  represents  a  mixture  of  colors  which  can 
be  easily  separated  as  Kraus173  has  indicated  into  a  green  and  a 
yellow  part. 

Add  to  an  alcoholic  extract  of  crude  chlorophyll  a  like 
quantity  of  benzole,  vigorously  shake  it  up  and  leave  the  mixt- 
ure a  short  time  to  itself,  and  the  alcohol  and  benzole  will 
again  separate.  The  under  fluid  is  now  a  yellow-colored  al- 
cohol, the  upper  a  green-colored  benzole.174  By  this  process 
the  crude  chlorophyll  is  separated  into  a  yellow  alcoholic  part, 
xanthophyll  (Kraus)  and  a  green  benzole  part,  kyanophyll 
(Kraus).  According  to  "\Viesner,175  instead  of  the  benzole  one 
may  use  fatty  oils  (linseed  oil,  nut,  poppy,  olive  oil)  essen- 
tial oils  (turpentine,  rosemary,  gaultheria  oil)  or  sulphuretted 
carbon. 

Kraus  and  others  therefore  held  that  the  yellow  coloring  matter 
(xanthophyll)  and  the  blue  green  (kyanophyll)  together  represent 
chlorophyll ;  that  they  are  both  components  of  the  same  green 
coloring  substance.  According  to  the  investigations  of  Pring- 
sheim  and  Wiesner  it  appears,  however,  that  the  kyanophyll 
of  Kraus  is  relatively  pure  chlorophyll,  but  that  the  xantho- 
phyll of  Kraus  consists  of  yellow  modifications  of  chlorophyll 

172  Kraus,  Chlorophyllfarbstofle,  vj.23. 

173  Kvaus,  I.  c.,  p.  87,  ff.    The  objections  raised  by  Konrad  (Flora,  1872,  p.  396,  /.)  rest  on 
insufficient  experiments  and  have  already  been   duly  confuted  by  Wiesnev  (Flora,  1874.  p. 
284,  /.)• 

174  Concerning  the  behavior  of  benzole  toward  alcohol  of  various  percentages,  see 
Pringsheim  in  Monatsber.  K.  Acad.  Berlin,  1874,  p.  648,  /. 

««  Wiesner  in  Flora,  1874,  p. 282,  /. 


412  THE  MICROSCOPE  IN  BOTANY. 

whose  relations  to  crude  chlorophyll  are  not  fully  established, 
but  which  as  such  occurs  independently  in  it. 

(a)  Benzole   Chlorophyll  (Kyanophyll,  Kraus).     The  chlo- 
rophyll procured  from  alcoholic  solution  of  crude  chlorophyll  by 
the  use  of  benzole  is  a  beautiful  green  with  a  distinct  shade  of 
blue.      It  has  a  strong  red  luster,  a  much  stronger,  more  car- 
mine red  luster  than  the  crude  chlorophyll  solution.     It  is  very 
sensitive  to  acids,  the  least  trace  being  sufficient  to  change  the 
beautiful   green  to    a   smutty  yellow   brown    or   bronze-green 
(Kraus).     If  the  separation  is  produced  by  sulphuretted  carbon 
or  the  above  named  fatty  or  essential  oils  the  solution  is  full 
green   and  has  a  strong  red    luster.     A   saturated  solution  of 
chlorophyll  in  pure  olive  oil  or  sulphuretted  carbon  is  a  deep, 
almost  a  black-green  color,  and  appears  dark-red  by  reflected 
diffused  daylight.     It  will  keep  a  long  time  in  the  light  if  the 
oxygen  is  excluded  from  it ;  also  in  the  dark,  even  if  oxygen  is 
admitted ;  but  by  the  admission  of  oxygen  to  it  in  the  light  it 
rapidly  loses  its  color  (Wiesner). 

(b)  The    Yellow,   Alcohol  Part    (JKantliopliyll,    Kraus)    is 
according  to  Kraus  a  pure  gold  yellow  and  shows  no  trace  of 
fluorescence  (also  Gerland  and  Rauwenhoff,  Filhol).     Accord- 
ing to  Pringsheim  it  has  a  touch  of  green  shade  and  is  distinctly 
fluorescent  if  one  examine  it  with  a  condensing  lens  in  direct 
sunlight.     Evaporated  to  dryness  there  remains  a  deep  yellow- 
brown,  sticky  hydroscopic  mass,  which  may  be  dissolved  again 
in   alcohol,    ether,    benzole  and  carbon    sulphate,  but   not   in 
water.     If  sulphuric  or  muriatic  acid  be  added  to  the  yellow 
solution  it  will  remain  yellow  for  a  short  time,  then   become 
emerald  green,  verdigris  green  and  finally  a  beautiful    indigo 
blue.     Organic  acids  do  not  apparently  alter  the  solution.      In 
the  sunlight  it  gradually  bleaches  out  after  several  days.     Ac- 
cording to  Kraus  the  gold-yellow  alcohol  solution  is  identical 
with  the  yellow  coloring  matter  of  etiolated  plants. 

According  to  the  investigations  of  Pringsheim176  and  Wiesner 
it  is  very  probable  that  the  yellow  alcohol  portion  of  a 
solution  of  crude  chlorophyll  is  a  mixture  of  one  or  more  yel- 

i7Q  Pringsheim  in  monthly  report  of  the  Imperial  Acad.  Berlin,  1874,  p.  628,  ff. 


THE  SPECTROSCOPIC  BEHAVIOR  OF  CHLOROPHYLL.       413 

low  modifications  of  chlorophyll  with  a  little  chlorophyll.  It 
first  appears  independently  in  the  crude  chlorophyll  and  is  no 
preexisting  component  of  it.  Pringsheim  investigated  three 
yellow  modifications  of  chlorophyll,  viz.,  etiolin,  xanthophyll 
and  anthoxanthin. 

Etiolin  is  the  coloring  matter  which  is  formed  by  etiolated 
growths  breathing  in  the  darkness.  It  is  a  yellow  modification 
of  chlorophyll  having  a  red  fluorescence,  soluble  in  alcohol,  ether, 
benzole,  and  carbon  sulphate  but  insoluble  in  water.  Its  solu- 
tion becomes  by  the  addition  of  muriatic  or  sulphuric  acid,  first 
verdigris  green  and  afterward  blue. 

Xanthophyll  (in  Pringsheim's  sense)  is  the  yellow  coloring 
matter  of  autumn  leaves.  It  behaves  towards  the  before  men- 
tioned dissolving  media  quite  like  etiolin,  but  becomes  emerald 
green  not  a  bine,  by  the  addition  of  muriatic  or  sulphuric  acid. 
The  yellow  coloring  matter  of  autumn  leaves  arises  from  a  pro- 
cess of  decomposition  in  the  crude  chlorophyll.  If  the  coloring 
mutter  of  the  yellow  xanthophyll  grains  be  extracted  by  alcohol 
the  grains  remain  behind  in  their  original  size.  These  are  but 
gradually  affected  by  concentrated  sulphuric  acid  but  boiling 
potash  changes  them  to  a  greasy  brown  mass.177 

Anthoxanthin  is  the  yellow  coloring  matter  of  yellow  flowers 
and  fruit.  It  will  be  described  further  on. 

Which  of  the  two  first  yellow  modifications  of  chlorophyll 
occurs  in  the  crude  chlorophyll,  whether  etiolin  or  xanthophyll, 
or  both  together,  cannot  be  previously  determined. 

B.     Spectroscopic  Behavior  of  Chlorophyll. 

The  optical  characteristics  of  a  solution  of  chlorophyll  have 
been  frequently  investigated,  since  Brewster  first  directed  atten- 
tion to  the  subject,  and  himself  observed  its  most  important 
phenomena,  which  led  him  to  views  that  proved  the  incorrect- 
ness of  some  of  the  statements  of  Newton  conceining  the  nature 
of  light.  Not  to  mention  that  he  first  observed  the  fluorescence 
of  a  solution  of  leaf  green,  he  discovered  its  dichromism  also, 

177  In  many  cases,  however,  before  the  appearance  of  the  xanthophyll  the  chlorophyll 
grains  pass  into  a  beautiful  green  amorphous  mass  (Sachs,  Flora,  1603,  p.  202.) 


414  THE  MICROSCOPE  IN  BOTANY. 

that  is,  the  quality  by  which  a  thin  layer  of  it  gives  an 
absorption  color  of  green  and  a/  thicker  one  of  red.  He  first 
also  observed  the  dispersive  power  of  chlorophyll  for  red  light 
(carefully  investigated,  later,  by  Stockes),  and  finally  the  pe- 
culiar absorption  spectrum  of  leaf  green.  Of  the  latter,  to 
which  we  here  exclusively  devote  our  attention,  special  and 
exact  investigations  were  subsequently  made  by  Askenasy, 
Kraus,  Pringsheim,  and  others.  It  has  been  shown  by  these 
naturalists  that  the  chlorophyll  spectrum  may  be  employed  under 
all  circumstances  for  the  recognition  pf  chlorophyll  and  its 
modifications,  that  also  a  spectro-analytic  investigation  of  chlo- 
rophyll is  possible,  which  has  been  much  employed  in  the  most 
important  studies  of  later  times. 


B  C         D  E  TJ 


j  100        \  200          300    \     \iOO        500,  600          700          800        \  900       1000 


VL          Vff 


The  absorption  spectrum  of  chlorophyll  shows  seven  dark 
bands,  which  correspond  to  the  places  of  maximum  absorption. 
The  seven  absorption  bands  are  counted  progressively  from  the 
red  towards  the  blue  and  are  designated  by  the  Roman  numerals 
I  to  VII.  I  to  IV  lie  in  the  anterior  part  of  the  spectrum  in 
the  region  of  least  refraction,  between  the  Fraunhofer  lines  A 
and  E  (bands  of  the  first  half  of  the  spectrum),  V  to  VII  in 
the  posterior  part  or  region  of  most  refraction  (bands  of  the 
second  half  of  the  spectrum).  Bands  I  to  IV  are  easily  per- 
ceived in  solutions  of  chlorophyll  of  medium  concentration. 
Bands  V  to  VII  often  give  a  continuous  absorption,  but  they 
always  appear  with  a  sufficiently  weak  concentration  of  the 
solution.  Fig.  140  represents  an  absorption  spectrum  with  all 


THE  SPECTROSCOPIC  BEHAVIOR  OF  CHLOROPHYLL.        415 

the  bands,  Fig.  141  a  spectrum  with  a  continuous  absorption  in 
place  of  bands  V  to  VII  (after  Kraus). 

The  visibility,  the  intensity,  the  relative  distance  apart  (with- 
in certain  limits)  of  the  band,  are  dependent  on  certain  different 
factors,  namely : — 

The  concentration  of  Ike  solution,  or  what  is  equivalent  the 
thickness  of  the  layer  of  coloring  matter — the  optical  concen- 
tration,—  conditions  the  number  and  the  intensity  of  the  bands, 
and  secondarily  also  their  position. 

The  dissolving  medium  conditions  at  the  •  same  time  lesser 
variations  iu  the  relative  distance  apart  of  the  bands,  as  well  as 
the  rapidity  or  tardiness  of  the  development  of  the  absorption. 

The  kind  of  chlorophyll  modification  has  no  influence  upon 
the  position  of  the  maximum  and  minimum  of  the  absorption, 


no.  Hi. 

but  rather  upon  the  slower  or  more  rapid  development  of  the 
absorption  within  the  single  absorption  bands. 

We  will  consider  next  the  spectrum  of  a  normal  alcoholic 
solution  of  chlorophyll.  The  anterior  half  of  the  spectrum  is 
produced  by  a  medium,  the  posterior  by  a  weaker  concentration 
of  the  solution.  Fig.  140  after  Kraus. 

Band  I,  deep  black,  both  edges  sharply  defined ;  lies  between 
the  Fraunhofer  lines  B  and  C  in  the  red. 

Band  II,  less  black,  very  dark  brown  however  abruptly  shad- 
ing out  toward  both  sides,  exactly  in  the  middle  between  C  and 
D  in  the  orange. 

Band  III,  for  the  most  part  much  less  dark  than  II,  penum- 
brated  toward  both  sides,  in  the  yellow  close  behind  the  sodium 
line  D.  Between  II  and  III  a  slight  lessening  of  the  light. 


416 


THE  MICROSCOPE  IN  BOTANY. 


Band  IV,  very  slender,  weak,  often  scarcely  visible  lying 
before  E  in  the  green,  the  green  behind  it  obscured. 

Band  V,  broader  than  I,  almost  black  in  the  middle,  both 
sides  shading  out,  lying  in  the  light  blue  portion  exactly  be- 
hind F. 

Band  VI,  broader  than  V,  almost  black  in  the  middle,  both 
sides  broadly  shading  out,  lying  in  the  indigo,  beginning  in  the 
middle  between  F  and  G  and  ending  at  G. 

Band  VII,  corresponding  to  the  whole  of  the  remaining  violet 
end  of  the  spectrum. 


FJG.  142. 


That  the  spectrum  of  the  alcoholic  solution  of  the  chlorophyll, 
here  described,  appertains  to  the  coloring  malter  as  such,  that 
the  chlorophyll  extract  has  not  been  subjected  to  a  fundamental 
decomposition  before  it  is  applied  to  the  investigation,  have  been 
demonstrated  by  Kraus,  by  the  fact  that  chlorophyll  within  the 
living  plant  produces  similar  or  identical  spectra. 

The  spectrum  of  a  single  chlorophyll  grain  (Fiir.  142)  has 
the  appearance  of  a  luminous  spectrum  through  which  a  dark 


33  C 


T> 


looo 


FIG. 143. 

line  is  drawn,  which  is  interrupted  in  the  red  and  yellow.  The 
darkening  between  B  C  corresponds  to  Band  I.  That  which 
begins  behind  b  and  runs  through  the  whole  posterior  part  of 
the  spectrum  corresponds  to  the  total  absorption  of  the  bands 

v-vn. 

The  spectrum  of  a  living  leaf  (Fig.  143  of  Deutzia  scabra 


THE  SPECTROSCOPIC  BEHAVIOR  OF  CHLOROPHYLL.   417 

after  Kraus)  is  not  essentially  unlike  the  spectrum  of  the  solu- 
tion. The  leaf  to  be  investigated  is  put  on  the  microscope 
stage  and  the  objective  shoved  down  till  it  touches  it.  By  suit- 
able magnification  one  may  now  recognize  bands  I  to  IV  very 
distinctly  not  altered  in  their  relative  position.  Band  V  is  also 
sharply  visible,  while  VI  and  VII  are  mingled  in  a  single  ab- 
sorption. If  a  double  layer  of  leaves  is  used,  band  VI  will  blend 
with  the  total  absorption  of  the  posterior  half  of  the  spectrum. 
If  an  alcoholic  solution  of  crude  chlorophyll,  of  whose 
spectrum  we  have  hitherto  been  speaking,  be  mixed  with  ben- 


B  C 


E  b 


i     n     in    iv 


FIG.  144. 


zole,  the  benzole  portion  (pure  chlorophyll,  kynnophyll  of 
Kraus)  will  give  a  spectrum  quite  like  the  other,  with  this  dif- 
ference that  the  relative  distances  apart  of  the  bands  and  the 
relative  breadth  of  the  same  will  have  undergone  some  alter- 
ation. Kraus  held  this  to  be  a  characteristic  of  his  kyanophyll, 
but  according  toPringsheim,  the  reason  for  it  is  to  be  sought  in 
the  different  influences  of  the  media  of  solution.178 

If  by  the  effect  of  light  or  oxygen  or  of  other  agents  (acids, 
etc.)  a  decomposition  of  the  chlorophyll  takes  place,  the  pro- 

"s  For  particulars  see  Pringsheim  (Monatsber.  Berl.  Acad.,  1S74,  p.  62S.  ff.}  especially 
also  the  simple  and  double  dividing  of  band  I  of  the  benzole  solution  of  chlorophyll  in 
certain  degrees  of  concentration. 

27 


418  THE  MICROSCOPE  IN  BOTANY. 

duct  of  the  decomposition  gives  a  very  different  spectrum  from 
that  of  the  original  coloring  matter  (see  concerning  this,  Kraus, 

I.e.,  p.  68, /I)- 

By  the  investigations  of  Pringsheim  it  has  been  established 
that  the  spectrum  of  chlorophyll  solutions  of  different  thick- 
nesses shows  certain  highly  characteristic  changes  which  may 
be  best  seen  in  Fig.  144,  copied  from  Pringsheim. 

The  horizontal  divisions,  a  to  i,  represent  the  spectrum  of  a 
single  degree  of  dilution  (or  thickness  of  layer)  of  an  alcoholic 
chlorophyll  solution  ;  a  is  a  layer  of  tlis  solution  10  mm.  thick,  i 
that  of  a  like  concentration  374  mm.  thick.  The  other  values  are 
apparent  from  the  illustration.  The  spectrum  is  divided,  accord- 
Ing  to  the  fundamental  scale  of  Sorby  and  Browning,  into  100 
or  1,000  parts.  B,  (7,  A  etc.,  give  the  position  of  the  Fraun- 
hofer  lines ;  I  to  VII  designate  the  absorption  bauds. 


1000 


FIG. 


The  spectrum  i  shows  the  bands  I  to  IV  very  clearly,  while 

V  to  VII  run  together  into  a  single  absorption.     The  spectra 
h,  <7,  /"are  like  this,  only  that  bands  I,  II  and  IV  become  more 
narrow,  and  the  absorption  of  the  second  half  'of  the  spectrum 
is  drawn  back  more  toward  F.      Spectrum  e  is  distinguished 
by  the  almost  total  disappearance  of  baud  III  and  the  coming 
out  distinctly  of  band  V.     In  d,  band  III  has  altogether  disap- 
peared, and  II   and  IV  become  almost  entirely  clear,  and  V, 

VI  and  VII  clearer.     In  c  of  the  anterior  bands  only  I  is  still 
to  be  clearly  seen ;  but  V,  VI  and  VII  are  coming  to  be  more 
distinctly  perceptible.     Finally,  in  b  and  a,  all  the  bands  but  I 
appear  no  more.     Baud  I  is  thus  the  most  persistent,  and,  ex- 
cept that   it  becomes  gradually  narrower  it  remains  quite  uu- 


THE  SPECTROSCOPIC  BEHAVIOR  OF  CHLOROPHYLL.        419 

changed.  It  has,  therefore,  a  special  importance  for  the  recog- 
nition of  very  dilute  or  much  modified  solutions  and  may  be 
designated  as  the  characteristic  chlorophyll  band. 

The  spectrum  of  etioliu  is  apparently  very  different  from  that 
of  chlorophyll  in  weaker  concentrations.  It  shows  no  absorp- 
tion bands  in  the  anterior  half  (Fig.  145, -after  Kraus),  while 
beyond  the  line  F  are  seen  three  absorption  bands  correspond- 
ing to  V,  VI  and  VII  of  the  chlorophyll  spectrum,  the  spaces 
between  which  are  shaded.  It  was  formerly  supposed  that 
the  coloring  matter  of  etiolized  plants  would  not  generally  pro- 


E  b 


iv 


FIG.  146. 


dace  the  bands  I  to  IV,  but  Pringsheim  has  shown  that  a  layer 
of  etiolin  sufficiently  thick  would  afford  a  spectrum  which  es- 
sentially agrees  with  that  of  chlorophyll  (Fig.  J46  is  con- 
structed in  the  same  manner  as  Fig.  144).  The  essential 
distinction  lies  in  this,  that  bands  I  to  IV  are  not  so  strongly 
pronounced,  and  appear  only  when  using  thicker  layers  of  the 
solution.  So  also  number  II  is  divided  into  two  bands,  a,  b, 
and  the  position  of  the  bands  in  the  blue  is  somewhat  altered. 
Etiolin  stands  therefore  optically  very  near  to  chlorophyll. 

The  spectrum  of  xanthophyll  in  Pringsheim's  sense  is  much 
more  variable.    It  shows  only  the  three  bands  in  the  blue,  and  it 


420  THE  MICROSCOPE  IN  BOTANY. 

is  still  uncertain  if  even  with  a  thickness  of  370  mm.  band  I  in 
the  red  really  exists.  But  if  the  solution  be  much  concentrated 
by  evaporation  and  then  tested  in  that  thickness,  there  appears 
quite  a  distinct  dark,  but  slender  band  I  from  the  lithium  line 
to  near  (7,  and  one  beginning  at  E  and  from  b  on  becoming  a 
very  dark  absorption.  On  the  other  hand  bands  II,  III  and 
IV  have  never  been  made  to  appear. 

The  absorption  appearances  in  the  spectrum,  especially  as  they 
are  produced  by  layers  of  fluid  of  different  thicknesses,  may  be 
graphically  expressed  by  the  form  of  curves,  the  so-called  ab- 
sorption curves.  Askenasy179  first  employed  them.  He  drew 
a  curve  from  the  spectrum  of  a  layer  whose  ordinates  stood  in 
relation  to  the  intensity  of  the  darkening,  so  that  the  maximum 
of  the  curve  corresponded  with  the  maximum  of  the  darkening. 

Without  photometric  apparatus  this  method  must,  however, 
lead  to  very  arbitrary,  or  at  least  subjective  results.  It  has, 
therefore,  been  but  little  used. 

Another  method  introduced  by  Pringsheim180  aims  at  the 
graphical  representation  of  the  maxima  and  minima  of  absorp- 
tion. It  brings  the  whole  course  of  the  absorption  before  the 
eye.  See  Figures  144  and  146. 

Theabscissa  axis  is  divided  into  100  or  1,000  parts,  correspond- 
ing to  the  scale  of  the  spectroscopic  measuring  apparatus,  when 
the  Browning  scale  is  the  standard,  or  directly  in  wave  lengths 
in  hundred  thousandth  parts  of  a  mm.  when  the  scale  of  Ang- 
strom181 is  the  standard.  The  ordinate  axis  gives  the  optical 
concentration  (height  of  fluid  layer  in  millimeters).  There  is 
obtained  in  this  way  a  coordinate  system  in  which  the  observed 
absorption  bands  may  be  directly  registered.  For  the  better 
locating  of  points,  the  position  of  the  Fraunhofer  lines,  B,  (7, 
Z),  JE,  b,  jp,  6r,  should  be  designated  above  the  abscissa  line, 

[Distribution  of  energy  in  the  chlorophyll  spectrum.] 

[C.  Timiriazeff*  points  out  the  intimate  relationship  between 
the  absorption  of  light  by  chlorophyll  and  the  intensity  of  the 

179  Askenasy  in  Botan.  Zeitg.,  1867,  Taf.  V. 

iso  Pringsheim  in  Monatsber.  d.  Berl.  Acad.,  1875,  p.  795. 

«i  Cf.  Nebelung  in  Bot  Zeitg.,  1872,  Taf.  XI. 

*  In  Comptes  Rendus,  I.  c.,  pp.  375-6. 


THE  COLORING  MATTER  OF  FLOWERS.  421 

chemical  phenomena  produced,  the  curves  of  absorption  of 
light  and  of  the  decomposition  of  carbonic  dioxide  presenting 
an  almost  exact  concurrence.  This  last  function  may  be  consid- 
ered as  dependent  on  the  energy  of  radiation,  as  measured  by 
its  effect  on  the  thermo-pilo.  Langley  has  definitely  fixed  the 
position  of  maximum  energy  in  the  solar  spectrum  to  be  in  the 
orange,  exactly  in  that  part  which  corresponds  to  the  character- 
istic band  of  chlorophyll  between  B  and  (7.] 

[It  follows,  therefore,  that  chlorophyll  maybe  regarded  as  an 
absorbent  specially  adapted  for  the  absorption  of  those  solar 
rays  which  have  the  greatest  energy,  and  its  elaboration  by  the 
vegetable  economy  is  one  of  the  most  striking  examples  of  the 
adaptation  of  organized  beings  to  the  conditions  of  their  en- 
vironment.] 

[Under  the  most  favorable  conditions  40  per  cent  of  the  solar 
energy,  corresponding  to  the  rays  of  light  absorbed  by  the 
characteristic  chlorophyll  bands  (see  pp.  153  and  413^*. )  is  trans- 
formed into  chemical  work.  Chlorophyll  therefore  constitutes 
an  apparatus  of  great  perfection  capable  of  transforming  into 
useful  work  40  per  cent  of  the  solar  energy  absorbed.] 

[Quoted  from  the  Journal  of  the  Royal  Microscopical  Society, 
Vol.  Ill,  No.  Ill,  p.  390.  A.  B.  H.] 

XL     THE  COLORING  MATTER  OF  FLOWERS. 

Literature.  Marquart,  Die  Farben  der  Bliiten,  Bonn,  1835. 
— Bohm,  Physiol.  Unters.  ii.  blaue  Passiflorabeeren  (Sitzungs. 
d.  K.  Acad.  d.  Wiss.,  Wien,  Bd.  XXIII,  1857,  p.  19,  /.)• 
— Wigand,  Einige  Satze  iiber  die  Bedeut.  d.  Gerbstoffe  u.  d. 
Pflanzenfarben  (Botan.  Zeitg.,  1862,  p.  121, /".). — Wiesner, 
Einige  Beobacht.  iiber  Gerb-  und  Farbstoffe  d.  Blumenbl.  (id. 
p.  389,  ff.). — Hildebrand,  Auat.  Unters.  iiber  d.  Farben  d. 
Bliiten  (Pringsheim's  Jahrb.,Bd.  Ill,  1863,  p.  59,/".). — Weiss, 
Unters.  iiber  d.  Entwicklungsgeschichte  d.  Farbstoffes  in 
Pflzellen  (Sitzungsber.  d.  K.  Acad.  d.  Wiss.  Wien,  Bd.  LIV, 
1  Abth.,  1866,  p.  157,/1.). — Nageli  u.  Schwendener,  Mikrosk., 
p.  500,  ff.—  Kraus,  Zur  Kenntn.  d.  Chlorophyllfarbstoffe,  etc., 
Stuttg.,  1872.— Kraus,  D.  Entsteh.  d.  Farbstoffkorper  in  den 


422  THE  MICROSCOPE  IN  BOTANY. 

Beeren  von  Solanum  Pseudocapsicum  (Pringsheim's  Jahrb., 
Bd.  VIII,  1872,  p.  131,  /*.).— Wiesncr,  Unters.  iiber  d.  Farb- 
stoffe  einiger  fiir  Chlorophyll frei  gehaltenen  Phanerog.  (id.  p. 
575,  ff.). — Pringsheim,  Ueber  d.  Absorptionsspectra  der  Chlo- 
rophyllfarbstoffe  (Monatsber.  d.  K.  Acad.  d.  Wiss.,  Berlin, 
187|,  p.  628,  ff.). — Pringsheim,  Ueber  natttrl.  Chlorophyll- 
mocnficationen,  etc.  (id.  1875,  p.  745,  ff.).—  Borscow,  Notiz 
iiber  d.  Polychro'ismus  einer  alkohol.  Cyaninlosung  (Bot.  Zeitg., 
1875,  p.  351).— Holstein,  D.  Schicksal  d.  Anthoxanthin- 
kornerin  abbliih.  Blumenkr.  (Bot.  Zeitg.,  1875,  p.  25,  ff.). 

Flahault,  Sur  la  form,  des  matieres  colorantes  dans  les  vege- 

taux  (Bull,  de  la  Soc.  bot.  de  France,  t.  XXVI,  1879,  p.  268, 

ff-)' 

The  coloring  matter  of  floral  leaves  and  colored  pericarps  is 

still  much  less  perfectly  known  than  chlorophyll  and  its  related 
substances.  Like  these  it  always  appears  as  a  part  of  the  cell 
contents,  never  united  with  the  membranes.  Either  it  is  dis- 
solved in  the  cell  sap  and  so  represents  a  fluid,  or  it  is  united 
with  variously  formed  granular  structures,  of  probably  proto- 
plasmic nature.  Dissolved  it  constitutes  chiefly  the  blue  violet 
and  rose  red  colors  ;  united  with  granules  it  is  yellow,  orange  and 
green.  To  both  cases  there  are,  however,  exceptions.  These 
colors  together  often  produce  mixed  colors.182  Since  the  in- 
vestigations by  Marquart  of  the  colors  of  flowers  we  desig- 
nate the  dissolved  blue  and  red  pigment  as  Anthocyan,183  the 
yellow  and  orange  colored  as  Antboxanthin.  Frerny  and  Cloez 
assumed  three  kinds  of  flower  coloring  matter,  viz.  Cyanin, 
the  blue  pigment,  probably  identical  with  Marquart's  Antho- 
cyan, red  coloring  matter  is  a  modification  of  it.  Secondly, 
Xanthein,  a  yellow  coloring  matter  soluble  in  water,  thirdly, 
Xanthin,  a  yellow  coloring  matter  insoluble  in  water,  which 
contains  a  considerable  quantity  of  fatty  matter  and  is  solu- 
ble* in  ether  and  alcohol.  Identical  with  Xanthein  is  probably 
the  Lutein  of  Thudichum  and  the  Anthochlor  of  Prantl.  Ac- 
cording to  Pringsheim,  the  separation  of  the  yellow  pigment  is 
inadmissible  both  being  according  to  him  Anthoxanthin,  a 

*82  For  this  see  Hildebrand,  I  c. 

183  The  red  might  also  be  especially  set  off  as  erythrophyll. 


THE  COLORING  MATTER  OF  FLOWERS.  423 

modification  of  chlorophyll  similar  to  etiolin  and  xanthophyll 
(see  p.  413,/.). 

A.  Anthocyan,   Cyanin,  soluble  in  alcohol  and  ether.     If 
the  dissolving  medium  be  evaporated,  the  blue  coloring  matter 
may  be  again  taken  up  with  water.     Acids  color  the  pigment 
red  or  violet,  alkalies  change  the  red  again  to  blue,  violet  or 
yellow  green.     According  to  Wiesner184  anthocyan  never  is  col- 
ored green  by  the  use  of  alkalies.     Where  this  is  the  case  the 
green  color  is  given  by  the  presence  of  tannin  which  is  colored 
yellow  with  alkali  and  gives  the  green  color  with  the  blue  an- 
thocyan   (Nageli   and   Schwendener,  and   Sachsse   controvert 
this). 

Spectroscopically  anthocyan  has  not  been  very  carefully  tested 
The  blue  modification  shows  an  absorption  beginning  at  D  and 
continuing  to  F,  the  violet  a  weak  one  at  />,  and  a  larger  one 
tit  the  blue  end  of  the  spectrum,  the  red  an  absorption  band  in 
green  and  blue  to  F,  and  an  end  absorption  beginning  at  G. 

B.  Anlhoxanthin  (including  xanthein,  lutein,  anthochlor). 
It  occurs  mostly  in  connection  with  proteid  substances  (rarely 
in  oily  substances).     It  is  not  distinguished  from  the  colorless 
fundamental  substance  of  the  chlorophyll  granules.     With  few 
exceptions  it  is  insoluble  in  water,  soluble  in  alcohol,  ether, 
benzole  and  other   media   for   dissolving   chlorophyll.     These 
solutions  are  colored  blue  with  acids  and  are  faintly  fluorescent. 
According  to  Hanstein  anthoxanthin  granules  are  changed  by 
the  fading  of  the  floral  crown  by  being  transformed  into  a  yel- 
low quite  homogeneous  mass. 

Pringsheim185  has  shown  that  the  spectrum  of  anthoxanthin 
itself  is  not  essentially  different  from  that  of  chlorophyll  (Fig. 
147).  An  alcoholic  solution  of  coloring  matter  in  thin  layers 
shows  only  bands  V,  VI,  VII,  which  soon  run  together  into  a 
continuous  terminal  absorption.  By  further  increasing  the  con- 
tents of  the  coloring  matter,  first,  band  I,  then  II  and  IV,  and  at 
last  baud  III,  make  their  appearance.  Anthoxanthin  shows 
spectroscopically  all  the  essential  marks  of  chlorophyll  and  is 
to  be  regarded  as  a  modification  of  it. 

is*  Wiesner  iti  Bot.  Zeitg.,  1862.  p.  389. 

iw  Monatsber.  d.  K.  Acad.,  Berlin,  1874,  p.  638,  ff. 


424 


THE  MICROSCOPE  IN  BOTANY. 


If  the  yellow  coloring  matter  of  the  flowers  occurs  as  a  solu- 
tion in  the  cells,  it  may  be  extracted  by  water,  and  colored 
brown  yellow  by  the  addition  of  potash  lye. 

Those  peculiar  coloring  substances  which  occur  in  pericarps 
and  elsewhere,  mostly  connected  with  granules,  and  which  have 
been  investigated  by  Bohm,  Weiss  and  others,  are  probably  to 
be  regarded  as  flower  pigments. 

The  blue  granules  in  the  fruit  of  the  Passiflora  are  soluble, 
according  to  Bohm,  in  water,  alcohol  and  ether,  both  cold  and 
boiling.  Potash  changes  their  color  to  yellow  brown.  Acids 
and  alkalies  dissolve  thorn. 


B  C 


II      III    IV 


FIG.  147. 


Weiss  has  given  the  reaction  of  numerous  granules  of  that 
kind,  the  most  important  of  which  are  the  following : — 

Orange  colored  granules.  Iodine  colors  them  green  (  Oucurbi- 
ta,  Aeschinanthus,  Canna,  Gazania,  Lilium,  Hemerocallis,  Cap- 
sicum) ,  or  blue  green  (  Gaillarda) ,  potash  does  not  alter  the  color 
(Cucurbita,  Aschinanthus,  Gazania)  but  dissolves  them,  some- 
times when  applied  dilute  (Aeschianthus) ,  sulphuric  acid  colors 
green-yellow  (Lilium),  nitric  acid  first  light  blue  then  the 
color  disappears  (Lilium). 

Yellow  granules.  Adonis:  iodine  does  not  alter  them,  like- 
wise benzole,  potash  bleaches  them  somewhat.  Tydaea:  iodine 
colors  yellow-green,  potash  is  without  effect. 


ASPAKAGIX.  425 

Carmine  red  granules.  Lycopersicum :  iodine  colors  them 
green  to  yellow-green,  potash  leaves  them  unchanged.  Col- 
umnea:  iodine  as  well  as  sulphuric  and  muriatic  acid  leaves  them 
unaltered,  but  coagulates  them.  Nitric  acid  makes  them  a 
vermillion  red  but  does  not  coagulate  them.  Chlorine  water  is 
without  effect. 

Violet  granules.  Convallaria :  iodine  colors  red,  acids  destroy 
the  color.  Solanum  melongena:  iodine  colors  gold  yellow, 
potash  blue. 

Blue  granules:  iodine  does  not  color  them,  but  potash  does 
a  beautiful  green  (Delphinium) . 

These  coloring  substances  arise  from  the  transformation  of 
chlorophyll.  Plasmic  forms  are  their  fundamental  substance. 
They  give  double  refraction. 


XII.     ASPAEAGIN. 

Literature.  Piria,  Rech.  sur  la  const,  chim.  de  1'asparagine, 
etc.  (Ann.  de  chim.  et  de  phys.,  3e  ser.,  t.  XXII,  1848,  p. 
160,  f.  ;  abgedruckt  aus  II  Cimento,  Jan.,  1846).  Pasteur, 
Noirvelles  rech.  sur  les  relations  qui  peuvent  exister  entre  la 
forme  cristalline,  la  comp.  chirn.  etc.,  (id.,  3e  ser.,  t.  XXXI, 
1851,  p.  70,  ff.) — Hartig,  Eutwicklungsgesch.  d.  Pflkeims., 
Leipzig,  1858,  p.  128,  /. — Pfeffer,  Unters.  fiber  d.  Protein- 
korner  u.  d.  Bedeut.  d.  Asparag.  beim  Keimen  d.  Samen 
(Pringsheim's  Jahrb.,  Bd.  VIII,  1872,  p.  530,  jf.).— Pfeffer, 
Ueber  d.  Bezieh.  d.  Lichtes  z.  Regen.  v.  Eiweissst.  aus  demb. 
Keiniungsprocess  gebild.  Asparagm  (Monatsber.  d.  K.  Acad. 
d.  Wiss.,  Berlin,  Dec.,  1873,  auch  Bot.  Zeitg.,  1874,  p.  249, 
ff. ;  ubersetz  in  Ann.  des  sc.  nat.,  5e  ser.,  t.  XIX,  1874,  p. 
371,- /I)— Pfeffer  in  Tagebl.  d.  46.  Yers.  dtsch.  Naturf.  u. 
Aerzte  z.  Wiesbaden,  Sect.  Bot.  (Bot.  Zeitg.,  1874,  p.  236). 
—Pfeffer,  D.  Bild.  stickstoffhalt.  Subst.  in  d.  Pfl.  (Jahrb.  f. 
Land  wirthsch.,  Bd.  Ill,  1873,  p.  437, /".).  Gorup-Besanez, 
AVeitere  Mitth.  liber  d.  Auftret.  v.  Leucin  neben  Asparagin, 
etc.  (Bot.  Zeitg.,  1874,  p.  379,  f.).  Borodin,  Ueber  d.  phy- 


426  THE  MICROSCOPE  IN  BOTANY. 

siol.  Rolle  u.  d.  Verbreit.  d.  Asparagins  imPflreiche  (id.,  1878, 

p.  801, jf.)-186 

Asparagin  (C4H8N2O3)  is,  it  would  seem,  a  widely  distrib- 
uted substance  in  the  vegetable  kingdom.  It  was  first  pre- 
pared from  shoots  of  asparagus  and  was  named  from  that  plant- 
It  is  found  in  the  milk-sap  of  the  sprouts,  stems,  root-tubers, 
fruit  and  seeds.  Examples  of  its  occurrence  are  furnished  by 
Convallaria,  Paris,  Ornithogalum  (roots,  weeds), germs, seeds, 
roots  and  the  stems  of  numerous  Papilionacece  which  have  been 
grown  in  the  dark,  potatoes,  althea  root,  seeds  of  Castanea, 
small  shoots  of  the  hop,  sprouts  of  Tilia,  Syringa,  Sambucus, 
Quercus  (Borodin).  Especially  in  the  germs  of  Lupinus  lu- 
teus  it  occurs  very  plentifully.  It  is  found  in  the  living  plant 
always  in  a  state  of  solution.  It  was  first  microscopically  ob- 
served by  Th.  Hartig  and  given  the  name  "gleiss"  (glister). 

In  respect  to  its  physiological  function  there  exist  two  oppo- 
site and  incompatible  theories.  According  to  one  (Pfeffer) 
asparagin  is  a  transitional  form  between  the  proteid-reserve 
substances  of  the  seed  and  the  living  albumen  of  the  developing 
plant  which  aids  in  the  transference  of  the  nitrogen.  It  is 
produced  from  proteid  substances  and  is  again  transformed  into 
them.  But  its  disappearance  stands  in  intimate  connection 
with  the  disappearance  of  sugar,  and  the  presence  of  that  is 
necessary  also  to  its  formation.  According  to  the  other  theory 
(Hartig,  Borodin)  asparagin  is  the  form  under  which  generally 
albuminous  substances  pass  from  cell  to  cell.  ("The  '  gleiss  ' 
crystal  is  to  a  certain  e'xtent  the  sugar  of  aleuron,"  Hartig). 
Proteids,  not  carbo-hydrates,  are  decomposed  in  the  formation  of 
asparagin  which  again  is  changed  back  into  albumen.  This  ex- 
plains the  fact  that  the  plant  is  always  poor  in  albuminous  sub- 
stances when  asparagin  occurs. 

Asparagin  crystallizes  easily  with  one  atom  of  water  in  limpid, 
transparent  orthorhombic  columns,  twin  forms  often  appearing. 
Fig.  148,  A^  JB,  represents  two  forms  of  asparagin  crystals 
which  have  been  many  times  observed  (macroscopic  according 
to  Pasteur)  (7,  D,  E,  F,  some  more  abundant  microscopic 
forms.  It  is  soluble  in  water,  acids  and  alkalies,  but  insoluble 

186  The  chemical  literature  in  Husemann,  1.  c.,  p.  261. 


ASPARAGIX. 


427 


in  absolute  alcohol  (not  in  dilute),  ether,  fatty  and  essen- 
tial oils. 

The  microscopical  'investigation  "should  be  conducted  with 
absolute  alcohol  (or  oil)  (Hartig,  Pfeffer  Borodin).  Add  a 
.drop  of  absolute  alcohol  to  a  section  lying  under  the  cover- 
glass.  Then  after  some  minutes  one  sees  crystals  shoot  out, 
partly  in  and  on  the  section,  partly  on  both  glasses  and  partly 
about  the  evaporating  edges  of  the  fluid.  The  crystals  may  be 
preserved  for  several  days  by  coyering  the  section  on  the  slide 
with  a  superficial  layer  of  oil.187 

In  order  to  test  the  presence  of  asparagin  in  the  cells,  take, 
according  to  Pfeffer,188  a  section  which  is  thicker  than  a  layer 
of  cells  and  lay  it  in  a  watch-glass  in  absolute  alcohol  and  move 
it  quickly  about.  Sections  with  little  asparagin  in  them  should 
be  put  on  a  slide  and  the  alcohol  added.  This  may  either  peiie- 


FIG.  US. 

trate  into  the  cells  too  quickly  and  so  prevent  the  outward 
diosmosis  of  the  asparagin,  or  it  may  be  too  dilute  in  the  neigh- 
borhood of  the  section  so  that  no  asparagin  can  then  be  separated 
out.  Alcohol  should  therefore  be  applied  to  the  preparation 

187  Hartig,  Entwickhingsgeschichte  cl.  Pflkeiras,  p.  127. 
iss  Pfeffer  in  Pringsheiru's  Jahrb.,  Bd.  VIII,  p.  533. 


428  THE  MICROSCOPE  IN  BOTANY. 

several  times.  According  to  Borodin189  this  is  not  a  suitable 
thing  to  do.  Alcohol  should  be  applied  to  the  section  but 
once  ;  lay  on  the  cover-glass  and  allow  it  to  dry.  In  this  way 
a  much  smaller  quantity  of  asparagin  may  be  detected  than  by 
adding  alcohol  several  times. 

Borodin190  gives  two  methods  for  the  more  certain  detec- 
tion of  crystallized  asparagin  as  such.  Warm  it  to  about 
100°  and  the  crystal  will  give  up  its  water  of  crystallization 
and  be  transformed  into  a  clear,  homogeneous,  strongly  refrac- 
tive drop,  which  has  an  outward  appearance  like  oil  and  which 
is  easily  soluble  in  water.  From  this  solution  it  may  again  be 
crystallized  by  means  of  alcohol.  But  if  it  be  heated  to  200° 
it  will  be  so  decomposed  that  it  appears  as  a  brown  drop  ap- 
parently filled  with  gas  bubbles  and  no  longer  soluble  in  water. 

But  one  may  dilute  the  crystallized  asparagin  with  a  satu- 
rated aqueous  solution  of  asparagin  which  is  not  colder  than  the 
object  to  be  tested.  It  is  best  to  take  a  cold  section  with  a 
slightly  warmed  saturated  solution  and  observe  under  the  micro- 
scope the  effect  of  a  drop  of  it  on  the  doubtful  crystals.  All 
other  crystals  soluble  in  water  will  dissolve  the  same  in  this  as 
in  pure  water,  but  asparagin  remains  unchanged. 

In  the  same  manner  Borodin  tested  tyrosiu  which  frequently 
occurs  in  company  with  asparagin. 


XIII.     INOKGANIC  VEGETABLE  ELEMENTS. 

Literature.  Raspail,  Mem.  de  la  Soc.  d'hist.  nat.  de  Paris, 
Sept.,  1828.— Sanioin  Monatsber.  d.  K.  Acad.  d.  Wiss.  Berlin, 
1857,  pp.  53,  ff.,  253,  ff. — Hanstein,  UeBer  ein  noch  nicht 
bekanntes  System  schlauchartiger  Getasse,  etc.  (id.  1859,  p. 
705,  ff.).— v.  Mohl,  Ueber  d.  Kieselskelett  lebender  Pflanzen 
(Botan.  Zeitg.,  1861,  No.  30,  ff.).— Wicke,  Ueber  d.  York, 
u.  d.  physiol.  Verwend.  d.  Kieselerde  (Bot.  Zeitg.,  1862,  p. 
76). — Sachs,  Ergebnisse  einiger  neuer  Unters.  iiber  d.  in  d. 
Pfl.  enth.  Kieselsaure,  I  (Flora,  1862,  p.  33,  ff.). — Sachs,  do., 
II  (id.  1883,  p.  113,  ff.).— Holzner,  Ueber  d.  Kryst.  in  d. 

189  Borodin,  I.  c.,  p.  804.  «o  Borodin,  I.  c.}  p.  805. 


INOKGANIC  VEGETABLE  ELEMENTS.  429 

Pflzellen  (Flora,  1864,  p.  273, /".).— Holzner,  Ueber  d.  phys- 
iol.  Bedeut.  d.  oxals.  Kalkes  (id.  1867,  p.  497, /I).—  Holzuer, 
D.  Krystalldrusen  in  d.  Bl.  d.  weissen  Maulbeerbaumes  (id. 
1867,  p.  470,/*.). — Rosanoff,  Ueber  Krystalldrusen  in  den  Pflan- 
zenzellen  (Bot.  Zeitg.,  1867,  p.  41,  ff.).— Hilgers,  Ucber  das 
Auftr.  der  Kiyst.  von  oxals.  Kalk  im  Pareuchym.  einiger  Mon- 
okot.  (Pringsheim's  Jahrb.,  Bd.  VI,  1867,  p.  285,  ff.).— Dip- 
pel,  D.  Mikroscop.,  Bd.  II,  1869,  p.  37,  /".—Graf  Solms- 
Laubach,  Ueber  einige  geformte  Vorkomann.  oxals.  Kalkes  in 
leb.  Zellmembranen  (Bot.  Zeitg.,  1871,  p.  509,  ff. ) .— Pfitzer, 
Ueber  d.  Einlagerung  v.  Kalkoxalatkryst  in  d.  pfl.  Zellhaut. 
(Flora,  1872,  p.  97,  ff.). — Vesque,  Obs.  sur  les  crist.  d'oxalate 
d.  chaux  dans  les  plants,  etc.  (Ann.  des  sc.  nat.  5e  ser.,  t. 
XIX,  1874,  p.  300,  f.).— Sacbs,  Lehrb.,  pp.  38-66.— Penzig, 
Z.  Verbreit.  d.  Cystolithen  im  Pflanzenreicb  (Bot.  Centrabl., 
Bd.  VIII,  1881  p.  393). — Also  numerous  statements  scattered 
through  various  treatises. 

The  inorganic  elements  occurring  in  plants  are  as  heteroge- 
neous as  they  are  wide  spread.  They  occur  in  every  cell  mem- 
brane, in  the  cell  contents,  cell  sap  and  protoplasm,  and  are  the 
indispensable  components  of  the  body  of  the  plant.  Their  pres- 
ence may  be  detected  by  the  incineration  of  a  portion  of  the 
plant,  after  which  process  they  remain  as  ashes,  often  indeed  very 
minute.  Commonly  they  are  not  to  be  recognized  by  means  of 
the  microscope.  More  seldom  they  appear  as  crystals  or  crys- 
talline forms,  or  also  as  amorphic  masses  in  the  cell  membrane  or 
cell  contents,  and  can  then  be  discovered  and  investigated  by 
means  of  the  microscope.  Only  the  latter  therefore  come  within 
the  province  of  our  inquiry. 

The  elements  having  an  inorganic  basis  which  are  visible  in 
the  cells  are  either  silicic  acid  or  lime  (or  magnesium  salts) 
What  physiological  role  they  play  is  indeed  a  question  often 
discussed,  but  as  yet  almost  altogether  unsolved.  Doubtless, 
indeed,  they  have  the  biological  function,  to  give  to  plants  and 
to  parts  of  plants  a  higher  degree  of  solidity  and  a  greater  re- 
sistance to  outside  influences,  especially  toward  the  assaults  of 
animals  (silex  layers  in  Equisetum  stems,  and  grass  blades,  cell 
walls  of  diatoms,  etc.).  The  salts  of  calcium  are,  on  the  con- 


430  THE  MICROSCOPE  IN  BOTANY. 

trary,  often  to  be  regarded  as  excretions  or  more  exactly  as  cell 
excrement. 

The  forms  to  be  described  here  are  insoluble  in  water,  but  in 
strong  mineral  acids,  mainly  muriatic  or  nitric  acids,  either  sol- 
uble or  insoluble.  Siliceous  secretions  are  insoluble  in  mineral 
acids,  but  calcium  salts  are  soluble  in  them. 

A.  /Silex.     It   occurs  in  the  cell  membranes  of  numerous 
plants,  in   the  stalk   and  leaves   of  many   grasses  and   Bam- 
busce,   in  the  sparkling  outer  layer  of  the   Catamites,   in   the 
epidermis  of  the  Equisetoe^  in  the  cell   walls  of  the  Bacillaria. 
It  is  insoluble  in  acids  and  alkalies,  and  is  incombustible,  and 
in  this  is  distinguished  from  every  other  vegetable  element  with 
an  inorganic  basis.     We  may  best  obtain  the  siliceous  incrusta- 
tion as  a  complete  skeleton,  by  calcining  the  part  on  the  plati- 
num slip,  after  having  first  withdrawn  the  other  inorganic  salts 
by  means  of  muriatic  or  nitric  acid  (for  method  see  p.  164). 

In  order  to  obtain  the  siliceous  frustules  of  diatoms  beautiful 
and  free  from  impurities  the  material  should  be  first  separated 
from  the  larger  impurities  by  a  fine  metal  sieve.191  Then  boil 
with  muriatic  acid  with  the  addition  of  calcium  chlorate,  where- 
by the  cell  contents  and  cellulose  membranes  will  be  destroyed 
and  the  frustules  will  be  separated.  The  mixture  is  then 
poured  with  a  considerable  quantity  of  water  into  a  high,  nar- 
row test-tube ;  let  it  settle,  pour  off  the  fluid  and  replace  it 
three  or  four  times  with  pure  water.  There  is  now  with  the 
diatom  frustules  a  small  quantity  of  impurity  in  the  form  of 
yellow  or  colorless  flakes  which  may  sometimes  be  removed  by 
boiling  the  material  in  water  to  which  is  added  a  piece  of  clean 
soap. 

B.  Calcium  salts.     By  far  the  most  frequently  occurring  in- 
organic element  belongs  to  the  calcium  salts,  and  indeed  the 
prevailing    forms  are  calcium  carbonate  and    calcium  oxalate, 
very  rarely  calcium  sulphate  or  phosphate  (see  p.  391). 

Calcium  oxalate,  which  forms  most  of  the  microscopic  crys- 
tals, crystallizes  in  quadratic  or  clinorhombic,  monoclinic  forms. 
Some  of  the  forms  of  crystals  most  frequently  found  are  repre- 
sented in  Fig.  149  (A,  B,  quadratic,  C-F,  monoclinic  forms). 

181  They  are  to  be  had  of  dealers  in  microscopic  objects. 


PLANT  SUBSTANCES  OF  LIMITED  DISTRIBUTION.        431 

When  the  frequently-occurring  monoclinic  pfisms  with  ortho- 
doms  are  very  much  developed  in  the  direction  of  the  longer  axis 
and  very  little  in  the  direction  of  the  transverse  axis,  the  formation 
of  crystalline  needles  takes  place  (raphides)  which  are  com- 
monly united  into  bundles,  the  needles  lying  parallel  and  near 
to  each  other  (bundles  of  raphides).  Aggregations  of  crystals 
of  calcium  oxalate  frequently  occur. 

Crystals   of  calcium   oxalate  are  insoluble  in  water,  potash 
lye  and  acetic  acid,  soluble  without  the  development  of  gas 


\r\ 

A 


FIG.  149. 

in  dilute  muriatic  acid.  If  the  crystals  have  been  previously 
calcined  they  dissolve  in  acetic  acid  with  the  formation  of  gas. 

Crystals  of  calcium  carbonate  are  soluble  in  dilute  acetic  and 
muriatic  acid  with  the  development  of  gas.  They  occur  now 
and  then  as  the  so-called  cystoliths. 

Crystals  of  calcium  sulphate  which  have  sometimes  been  ob- 
served are  soluble  in  cold  water. 


B.     PLANT  SUBSTANCES  OF  LIMITED  DIS- 
TRIBUTION. 

Few  of  the  vegetable  substances  of  limited  distribution  were 
at  first  included  in  the  domain  of  microscopical  analysis.  Whole 
groups,  as  for  example,  of  the  most  important  chemical  as  well 
as  technical  vegetable  bases,  are,  microscopically,  almost  totally 
unknown.  Others  have  indeed  been  better  studied,  but  in 
respect  to  these  also  many  questions  still  remain  unsolved. 


432  THE  MICROSCOPE  IN  BOTANY. 

The  substances  described  below  are  separated  into  groups 
which  (up  to  the  last  one)  correspond  to  their  chemical  be- 
havior. They  are  therefore  directly  connected  with  those  of 
the  preceding  section.  They  are  :  1  Glycoside,  2  Tannic  acids, 
3  Alkaloids,  4  Fatty  oils,  5  Essential  oils,  6  Stearoptine,  7 
Resin,  8  Phanerogamic  coloring  matter,  9  Cryptogamic 
coloring  matter. 


XIV.     GLYCOSIDE. 

Literature.  Hartig,  Ueber  d.  Zucker  u.  einem  dem  Saliciu 
ah nl.  Korper  aus  d.  Cambiumsafte  der  Nadelholzer  (Bot.  Zeitg., 
1863,  p.  413,  /.). — Nageli  und  Schwendener,  Mikrosk.,  p. 
494^  ft — Franchimont,  Rech.  s.  1'origine  et  la  const,  chim.  des 
resines  de  terpenes  (Arch,  neerland,  t.  VI,  1871,  p.  426,  ff.). 
— Tiemann  u.  Harmann,  Ueber  d.  Coniferin,  etc.  (Ber.  Deutsch. 
Chem.  Gesellsch.,  Bd.  VII,  1874,  p.  608 ,/".). -Tangl ,  Vor- 
lauf.  Mitth.  liber  d.  Verbreitung  d.  Coniferins  (Flora,  1874,  p. 
239, /".).— Miiller,  Ueber  Coniferin  (id.  p.  399.)— Borscow, 
Beitrage  z.  Histochemie  der  Pfl.  (Bot.  Zeitg.,  1874,  p.17,/1). 
— Pfeifer,  Hesperidin,  e.  Bestandth.  einiger  Hesperideen  (id. 
p.  529,  ff.). — v.  Hohnel,  Ueber  d.  Kork  u.  verkorkte  Gewebe 
iiberhaupt  (Sitzungsber.  d.  K.  Acad.  d.  Wiss.  Wien,  Bd. 
LXXVII,  1  Abth.,  1877,  p.  700,^.).— v.  Hohnel,  Histocheni. 
Unters.  liber  d.  Xylophilin  u.  d.  Coniferin  (id.  p.  699,  ff.). — 
Schwartz,  Chem-botan.  Studien  liber  d.  in  den  Flechten  vor- 
komm.  Flechtensauren  (Cohn's  Beitrag.  z.  Biologic  d.  PH., 
Bd.  Ill,  1880,  p.  249,  /I).— Singer,  Beitr.  z.  nalieien  Kennt- 
niss  d.  Holzsubstanz  u.  verholzt.  Gewebe  (Sitzungsber.  d.  K. 
Acad.  d.  Wiss.  Wien,  Bd.  LXXXV,  1  Abth.',  1882,  p. 
347,  J.). 

Under  this  designation  is  included  that  series  of  vegetable 
substances  which  are  produced  by  the  action  of  dilute  alkalies 
or  acids  on  cane  and  grape  sugar  and  their  relatives  (not  includ- 
ing others  produced  therefrom  by  dividing  the  sugar  molecule) . 
Most  of  these  are  in  a  pure  state  crystallized  and  soluble  in 
water ;  many  also  in  alcohol ;  others  are  insoluble  in  the  latter 


GLYCOSIDES.  433 

and  may  be  separated  by  means  of  this.  Of  the  numerous  bodies 
belonging  to  this  group  the  microscopical  investigation  of  the 
following  has  been  attempted:  Coniferin  (abietin),  vanillin, 
salicin,  hesperidin,  frangulin,  syringin  and  chrysophonic  acid 
(of  the  latter  it  is  still  doubtful  if  it  belongs  to  this  group). 
Most  of  the  following  statements  require  still  further  verifi- 
cation. 

1.     CONIFERIN  (Abietin)     C]6  H^CV 

It  crystallizes  in  white  or  yellow  needles,  soluble  in  water, 
not  easily  soluble  in  alcohol  and  ether.  With  concentrated 
sulphuric  acid  it  gives  a  violet-tolue  color  which  by  the  subse- 
quent addition  of  water  becomes  blue.  According  to  Franchi- 
mont,  coniferin  can  be  detected  in  the  cells  by  means  of 
sulphuric  acid,  giving  them  a  purple-violet  coloring.  It  be- 
comes green  with  muriated  carbolic  acid  (more  particularly  as 
to  this  substance  as  well  as  its  occurrence  in  lignified  membrane, 
see  pp.  340-1,  /*.). 

Hartig's  abietin  is  probably  identical  with  this.  It  is  with 
difficulty  soluble  in  water  and  ether,  easily  soluble  in  dilute  al- 
cohol. It  occurs  in  the  cambian  sap  of  many  Conifer  ce  and  can 
be  detected  by  treating  a  section  of  this  wood  with  concentrated 
sulphuric  acid.  It  shows  itself  in  a  characteristic  violet  blue 
color  in  the  whole  region  of  the  bast  ring.192 

2.    VANILLIN.    C8H8O3 

White  crystal  needles  soluble  in  much  water,  alcohol  and 
ether,  becoming  yellow  with  concentrated  sulphuric  acid  (with 
iron  chloride,  dark  violet).  According  to  Singer  it  constantly 
occurs  in  lignified  cell  membranes  and  affords  the  well-known 
reaction  oi  wood  substance  (see  p. 


3.    SALICIN.    C13H18O7. 
Likewise  crystallizes  (orthorhombic)  ;  insoluble  in  ether,  sol- 

»M  Hartig  in  Bot.  Zeitg.,  1863,  p.  414. 


28 


434  THE  MICROSCOPE  IN  BOTANY. 

<uble  in  water  and  alcohol,  still  more  easily  in  potash  water  as 
well  as  in  acetic  acid.  It  occurs  in  the  bark  of  many  species  of 
fSalix  and  Pqpulus.  With  concentrated  sulphuric  acid  it  is  col- 
ored a  beautiful  red  (with  the  addition  of  water  the  color  is  re- 
moved with  the  formation  of  a  red  powdery  precipitate  :  Rutilin) . 


4.    HESPEKIDIN.    C^Hg 

A  gtycoside  in  the  ripe  and  unripe  fruit  and  other  parts  of 
the  orange  tree.  It  is  insoluble  in  water  and  in  dilute  acids, 
.easily  soluble  in  potash.  It  occurs  in  the  cells  in  a  state  of 
solution,  and  after  lying  in  alcohol  it  is  precipitated  in  the  cells 
in  the  form  of  spherical  crystals  (also  in  glycerine),  which  dis- 
solve in  alkalies  with  a  yellow  or  reddish  color. 


5.     PHANQULIN     (RhamnoxantJiin)  C^H^Ou  (?). 

This  occurs  in  the  peripheral  portion  of  the  pith  of  Khamnus 
frangula,  also  in  the  woody  parenchyma  of  the  pith,  in  the  thin 
walled  phloem  elements  and  in  the  bast  parenchyma.  Frangulin 
is  a  crystal lizable  body  easily  soluble  in  alcohol.  In  the  cells  are 
\very  small  starch  grains  which  bear  the  frungulin,  and  which 
give,  with  iodine,  the  characteristic  blue  color.  The  grains 
•color  with  ammonia  or  potash  lye  a  beautiful  blood  red. 


6. 

Crystallizes  in  fine  white  needles  which  have  a  kind  of  silky 
sparkle.  Easily  dissolves  in  concentrated  sulphuric  acid  where- 
by the  solution  first  becomes  yellow  green,  then  blue,  and  at 
last  violet  red.  This  quality  is  very  useful  in  microscopically 
testing  syringin.  To  the  transverse  or  longitudinal  section  of 
the  twig  of  Syringa  vulgar  is  (in  which  syringin  occurs)  on  the 
slide,  add  relatively  concentrated  sulphuric  acid  (one  drop  H2SO4 
and  two  drops  H2O). 

As  soon  as  the  section  is  penetrated  by  this  solution  imme- 
diately the  entire  cell  wall  of  the  wood,  bast  and  medullary  ray 


TAXNIN.  435 

cells  are  colored  a  yellow  green ;  after  a  few  minutes  this  color 
ia  over  into  blue  and  finally  into  violet  red.  The  cell  rneni- 
branes  of  the  rest  of  the  tissue  as  well  as  the  cell  contents  remain 
quite  colorless.  By  the  use  of  still  more  dilute  acid,  however, 
the  reaction  occurs,  more  gradually  (after  two  or  three  hours). 
Further,  the  age  of  the  cell  membranes  is  not  a  matter  of  indiff- 
erence ;  the  younger  take  the  color  much  more  rapidly  than  the 
older  ones.  The  reaction  produced  by  the  acid  soon  becomes 
indistinct,  in  that  by  a  subsequent  apparent  diffusion  it  soon 
also  colors  the  cell  contents  red  violet.  Syringin  occurs  in  the 
cell  walls  of  thick  walled  phloem,  xyleni  and  xylem-medui- 
lary-ray  cells  (Borscow) . 

7.    CHRYSOPHANIC  ACID     (Rhein)  Ci5H10O4. 

It  crystallizes  in  beautiful  orange  yellow  needles  which  spar- 
kle like  gold.  It  is  scarcely  soluble  in  cold  water,  but  is  soluble, 
on  the  contrary,  in  ether  and  benzole.  Alkalies  produce  with 
it  in  solution  a  beautiful  purple  red  color.  It  is  found  in  the 
thalus  of  several  lichens  as  well  as  in  roots  of  Rliabarber,  Ru- 
mex  obtusifolius,  R.  patientia  and  in  Cassia  bijuga  (rind) .  In 
the  cells  it  is  connected  with  small  plasma  grains  which  take  on 
a  dark  purple  red  color  with  ammonia.  In  the  young  lateral 
roots  of  Rumex  obtusifolius  it  occurs  in  the  parenchyma  of  the 
outer  rind,  in  the  thin- walled  phloem  elements  as  well  as  in 
the  thin- walled  parenchyma  cells.  In  the  older  lateral  roots 
the  rind  parenchyma  is  quite  free  from  chrysophanic  acid,  but 
it  occurs,  on  the  contrary,  in  the  parenchyma  of  the  pith  (Bor- 
scow). 

XV.     TAXXIC  ACID  (TAXXIX). 

Literature.  Sachs,  Ueber  einige  neue  mikrosk.-chem.  Reac- 
tionsmeth.,  II  Ueber  mikrosk.  X^achweisung  d.  Gerbst.  in  d. 
Zellen  (Sitzungsber.  d.  K.  Acad.  d.  TTiss.  Wein.,  Bd.  XXXVI, 
1859,  p.  23  ff.). — Sanio,  Einige  Beinerk.  iieber  d.  Ban  d. 
Holzes  ;  V,  Ueber  Gerbstoff  (Bot.  Zeitg.,  p.  213  f.).— Wigand, 
Einige  Satze  iiber  d.  physiol.  Bedeut.  d.  Gerbstoffes,  etc.  (id., 
1862,  p.  121  ff). — Wiesner,  Einige  Beobacht.  iiber  Gerb-.  und 


436  THE  MICROSCOPE  IN  BOTAOT. 

Farbst.  d.  Blumenbl.  (id.,  p.  389/1). — Sanio,  Einige  Bemerk. 
iiber  den  Gerbstoff  u.  seine  Verbreitung  b.  d.  Holzpfl.  (id., 
1863,  p.  17,/.).— Hartig,  Ueber  d.  Gerbmehl  (id.,  1865,  Nr. 
7.), — Nageliu.  Schwendener,D.  Mikrosk.,p.  490/". — Hanstein, 
Ueber  d.  Org.  d.  Harz  und  Schleimabsonderung  in  d.  Laubkn. 
(Bot.  Zeitg.,  1868,  p.  721/.).—  Dippel,  DasMikrosk.,  Bd.  II, 
p.  20. 

Under  this  name  we  include  a  series  of  chemical  combinations 
which,  like  the  carbo-hydrates,  consist  of  carbon,  oxygen,  and 
hydrogen,  but  which  are  much  richer  than  they  in  carbon  and 
oxygen.  They  are  still  but  little  known  as  to  their  chemical 
constitution. 

Tannic  acid  occurs  especially  in  woody  growths  and  perennial 
herbs,  rarely  in  annual  plants,  more  frequently  in  dicotyledons 
than  in  monocotyledons.  Whole  families  of  plants  lack  tannin 
as  Solanece,  Oleacece.  On  the  contrary,  the  representatives  of 
the  Cupulifereoe,  Ericaceae,  Leguminosece  and  Romceas  are  es- 
pecially rich  in  them.  Tannic  acid  is  found  chiefly  in  the  bark, 
in  young  wood,  in  the  thin  walled  vascular  bundles,  rarely  in 
the  pith. 

Originally  tannic  acids  always  occur  dissolved  in  the  cell  sap, 
but  may,  in  later  stages,  penetrate  and  permeate  the  cell  wall. 
They  appear  also  to  pass  from  cell  to  cell.  Another  kind  of 
tannic  acids  (bark  of  the  oak,  etc.)  is,  however  not  capable  of 
diosmosis.  Their  further  progress  comes  about  only  when  they 
shall  have  been  transformed  into  other  products.  According  to 
Wigand  there  exists  a  certain  connection  between  tannin  and 
starch.  It  often  occurs  in  great  abundance  in  the  pulp  of  fruit, 
but  it  subsequently  disappears  in  the  mass  as  the  sugar  increases 
so  that  a  transformation  of  tannin  into  sugar  seems  to  take  place. 
In  other  cases  the  tannins  are  to  be  regarded193  as  chromogens 
out  of  which  the  blue  and  red  colors  of  flowers  are  subsequently 
developed. 

The  regular  appearance  of  tannic  acids  in  certain  pathological 
cell  growths  (gall  apples)  is  well  known. 

All  tannic  acids  are  soluble  in  water  and  in  alcohol  and  have 
an  astringent  taste. 

103  Wigand  in  Botan.  Zeitg.,  1862, 1.  c. 


TANNIN.  437 

There  are  some  very  characteristic  microscopical  reactions  for 
the  tannic  acids  by  which  they  may  be  easily  and  safely  recog- 
nized. 

With  iron  salts,  ferric  acetate,  ferric  vitriol,  ferric  chloride, 
and  others,  tannin  gives  a  dark  blue  or  green  color  or  even  a 
precipitate  with  these  colors.  Formerly  it  was  thought  that  the 
blue  and  green  shades  were  produced  by  different  kinds  of  tan- 
nin, and  they  were  therefore  divided  into  iron  green,  and  iron- 
blue  (both  are  sometimes  found  united  in  the  same  cell). 
According  to  the  later  investigations  of  the  "chemist,  however, 
there  are  no  sufficient  grounds  for  this  division.  It  is  best  to 
employ  in  the  reactions  a  not  too  concentrated  solution  of  the 
iron  salt.  Lay  the  section  immediately  into  it  and  examine  it 
at  once.  Or  one  may  bring  the  resigent  to  the  section  as  it  lies 
in  glycerine.  One  should  work  quickly  in  order  to  prevent  the 
tannin  solution  from  passing  by  diosmosis,  out  of  the  cells  into 
the  water  used  in  the  preparation,  and  so  make  the  reaction 
indistinct.  In  the  freshly  prepared  specimen  the  cell  membrane 
commonly  remains  colorless,  but  if  it  have  already  remained  a 
long  time  in  water  the  cell  membrane  will  be  colored  on  account 
of  the  tannin  having  penetrated  it.  In  many  cells  the  tannin 
mass  has  an  oily  appearance,  and  separates  by  the  addition 
of  water  into  small  globules  (bark  of  oak,  poplar).  The  blue 
color  produced  by  the  iron  chloride  assumes  here  at  last  a  brown 
shade,  from  which  we  infer  that  along  with  the  iron-bluing  tan- 
nin there  occurs  some  other  substance,  or  another  less  sensitive 
tannin  in  the  oily  masses  just  mentioned.19* 

A  second  reagent  for  tannic  acid  is  according  to  Sachs193  potas- 
sium hydroxide.  It  produces  an  oxidation  product  of  tannin 
which  is  a  brick  red,  yellow  red  or  red  brown  fluid.  This, 
like  the  precipitate  of  iron  salts,  is  clearly  visible  even  in  very 
thin  sections  and  with  the  presence  of  very  little  tannin.  Natur- 
ally here  and  there  sections  used  for  investigations  are  more  than 
a  single  layer  of  cells  thick.  Since  the  red  coloring  with  potash 
rests  upon  an  oxidation  process  one  must  commonly  wait  for 
several  minutes  for  the  reaction  to  take  place.  Lay  the  slide 

19*  Xageli  u.  Schwendener,  1.  c.,  p.  492. 
"5  Sachs,  I.  c.,p.27,ff. 


438  THE  MICROSCOPE  IN  BOTANY. 

with  the  section  on  white  paper  and  cover  the  latter  with  a  drop 
of  strong  potash  solution.  In  order  to  convey  air  necessary 
to  the  oxidation  of  the  tannin  cells  a  few  drops  of  water  should 
be  added.  However,  without  this,  the  coloring  takes  place 
though  much  more  gradually. 

In  potassium  iodide  of  iodine,  tannin  takes  on  a  yellow  or  yel- 
low brown  color,  with  dilute  chlor- iodide  of  zinc  solution  there 
appears  a  reddish,  rose  red  or  red  brown,  or  even  a  violet  pre- 
cipitate.196 These  colors  are  distinctly  recognizable  in  the  pres- 
ence of  a  very  little  tannin. 

A  solution  of  potassium  bichromate  colors  tannin  dark  red  or 
red-brown.  This  red-brown  combination  does  not  dissolve  in 
an  excess  of  the  reagent.  Ssmio197  laid  fragments  of  the  branches 
which  had  been  previously  dried  for  a  few  hours,  in  the  dilute 
reagent  and  prepared  the  sections  from  these  when  they  had 
become  thoroughly  penetrated  with  the  fluid.  But  the  section 
can  be  impregnated  with  the  salt  on  the  slide. 

Hansteiu's  aniline  solution  colors  tannin  roe-brown  in  bright 
or  strong  shades  (Hanstein). 

Gallic  acid,  according  to  Sachs,  will  give  with  baryta  solution 
a  gray  blue  precipitate ;  with  chlor-iodide  of  zinc  it  gives  a  rose 
red  precipitate. 

XVI.     ALKALOIDS. 

Literature.  Borscow,  Bcitrage.  z.  Histochemie  der  Pfl.  (Bot. 
Zeitg.,  1874,  p.  11  ff.). 

The  attempt  is  now  made  for  the  first  time  to  subject  to  a 
microscopical  test  a  substance,  belonging  to  a  group  of  vege- 
table bases,  which  the  chemist  has  very  carefully  studied,  viz.  : 

Vei  atrum     C32  H62  N2  O8. 

The  alkaloid  of  Ver atrum  album  and  V.  sabadilla  is  a  white 
crystalline  powder  which  is  insoluble  in  water  but  soluble  in 
alcohol,  ether,  chloroform,  benzole  and  glycerine  (with  diffi- 
culty) .  Concentrated  sulphuric  acid  dissolves  it  with  a  yellow 

196  Sanio  in  Bot.  Zeitg.,  1863,  p.  211. 
"7  Sanio,  idem,  p.  17. 


FATS.  439 

color,  this  soon  becomes  orange,  then  blood  red,  finally  carmine 
red  or  smutty  violet.  Borscow198  makes  use  of  fine  transverse 
and  longitudinal  sections  to  which  he  adds  sulphuric  acid,  which 
contains  a  double  volume  of  water  in  order  not  to  destroy  the 
delicate  tissue.  Then  the  characteristic  color  and  change  of 
color  appear.  The  under-ground  parts  of  the  Veratrum  were 
tested  in  this  manner,  and  it  was  found  that  in  the  roots  and 
in  the  continuation  of  the  axis  beneath  the  bulb,  is  the  principal 
seat  of  the  alkaloid  in  the  elements  of  the  epidermis  and  the 
protecting  layer,  and  that  in  the  scales  of  the  bulb  only  the  epi- 
dermal layer  contained  a  litlle  veratrum.  The  veratrum  appears 
to  occur  principally  in  the  inside  of  the  cell  wall. 


XVII.     FATS. 

Literature.  Karsten,  Ueber  d.  Ensteh.  d.  Harzes,  AYachses, 
etc.,  durch  d.  assimil.  Thatigkeit  d.  Pflzellen  (Bot.  Zeit.,  1857, 
p.  313,/.).—  Kutzing,  Grundz.  d.  philos.  Bot.,Bd.  I,  a.  v.  O. 
— Sachs,  Ueber  d.  Auftreten  d.  Starke  bei  d.  Keimung  olhaltiger 
Samen  (Bot.  Zeitg.,  1859,  p.  177, /".).— Sachs,  Ueber  d.  Stoffe, 
welched.  Material  z.  Aufbau  d.  Zellhaute  liefern  (Pringsheim's 
Jahrb.,  Bd.  Ill,  1863,  p.  183, /".).— Wigand,  Ueber  d.  Des- 
organisation  d.  Ptianzelle,  etc.  (Pringsheim's  Jahrb.,  Bd.  Ill, 
1803,  p.  155,  jf.).—  Miiller,  Unters.  iiberd.  Vertheil.  d.  Harze, 
etc.  in  Pflanzenkorper  (id.,  Bd.  V,  1866,  p.  387,  ff.). — Uloth, 
TVachsbildung  im  Pflanzenreich  (Flora,  1867,  p.  385,  ff.) — 
De  Bary,  Ueber  d.  AVachsuberziige  d.  Epidermis  (Bot.  Zeitg., 
1871,  p.  128,  jf.).— Pfeffer,  Unters.  iiberd.  Proteinkorner,  etc. 
(Pringsheim's  Jahrb.,  Bd.VIII,  1872,  p.  419, /".).—  Pfeffer,  D. 
Oelkorper  d.  Lebermoose  (Flora,  1874,  p.  2,^*.). — TTiesncr, 
Ueber  d.  Krystallin.  Bcschaffenheit  der  geformten  Wachsub- 
erzuge  pflanzl.  Oberhaute  (Bot.,  Zeitg.,  1876,  p.  225,^".). 

The  fats  are  diffused  vegetable  substances  of  a  peculiar  ap- 
pearance. They  are  either  fluid  (fatty  oils)  or  they  are  solid 
bodies  (wax).  The  fatty  oils  will  in  most  cases  represent  re- 
serve substances.  They  occur  therefore  in  a  great  number  of 

«3  Borscow,  1.  c.,  p.  38,  JT. 


440  THE  MICROSCOPE  IN   BOTANY. 

resting  seeds  (Sachs,  Pfeffer,  see  p.  382).  The  waxes  seem 
never  to  be  reserve  material,  but  they  appear  rather  in  a  num- 
ber of  cases  to  have  certain  biological  functions.  Where  they 
cover  the  surface  of  growths  they  prevent  the  penetration  of 
dampness  since  they  are  impervious  to  water.  That  the  cuti- 
cle is  often  covered  and  permeated  with  wax  has  been  shown 
by  De  Bary.199 

1.    PATTY  OILS. 

Fatty  oils  occur  in  the  cells  in  the  form  of  globular  drops 
which  are  easily  recognized  on  account  of  their  refractive  qual- 
ities. They  are  insoluble  in  water,  somewhat  soluble  in  alcohol, 
easily  soluble  in  ether,  benzole,  sulphurated  carbon,  and  acetic 
acid.  All  alkalies  destroy  them  by  saponification  (formation 
of  alkali  salts). 

Osmic  acid  and  alcanna  tincture  are  the  principal  microscop- 
ical tests  for  fatty  oils. 

Osmic  acid  colors  the  drops  of  fatty  oils  a  deep  brown  or 
black  brown,  alcanna  tincture  a  beautiful  red  (for  method  see 
pp.  310  and  385).  In  order  to  distinguish  the  drops  colored 
red  by  the  alcanna  tincture  from  resin  drops  or  drops  of  essen- 
tial oil  (which  are  colored  by  it  in  the  same  way),  add  a  little 
absolute  alcohol,  in  which  if  the  drops  are  not  dissolved,  they 
are  shown  to  be  fatty  oil. 

Concerning  the  somewhat  variable  behavior  of  the  oil  drops 
of  the  Hepaticce  toward  dissolving  media  see  Pfeffer  in  Flora, 
1874,  p.  2,  ff. 

2.    WAX 

Wax  occurs  as  a  solid  whitish  or  yellowish,  sometimes  crys- 
talline crust  on  the  surface  of  stems  and  leaves.  (Acer,  Cerox- 
ylon  andicola,  Mi/rica  cerifera,  Klopstockia,  Liriodendron 
tulipifera,  Eucalyptus,  Acacia  cultriformis,  etc.)  and  on  the 
fruit  of  many  plants,  as  the  so-called  "rime."  It  is  insoluble 
in  water,  a  little  soluble  in  cold  alcohol,  soluble  in  boiling  alco- 
hol, ether,  chloroform  and  essential  oils.  Alkalies  and  acids 

199  De  Bary  in  Bot.  Zeitg.,  1871, 1.  c. 


ESSENTIAL  OILS.    CAMPHOR.  441 

change  it  scarcely  at  all.  It  often  appears  to  arise  from  a  met- 
amorphosis of  cellulose.  Iodine  and  sulphuric  acid  do  not  color 
it,  or  if  at  all,  slightly  yellowish. 

There  are,  as  yet,  no  microscopical  methods  of  reaction  on 
wax. 

XVIII.     ESSENTIAL  OILS. 

Literature.  Elsewhere  given ;  partly  with  the  fats,  partly 
with  the  resins. 

Colorless,  yellow,  red,  brown  or  otherwise  colored,  strongly 
refractive  fluids  within  the  cells,  altogether  filling  them  or  form- 
ing single  drops  in  the  cell  sap,  perhaps  standing  in  certain 
relations  to  the  resins.  They  are  but  slightly  soluble  in  water 
but  easily  soluble  in  alcohol,  especially  in  absolute  alcohol  as 
well  as  in  ether.  With  sulphuric  acid  they  commonly  take  on 
a  brown  coloring. 

They  may  be  microscopically  tested  by  alcanna  tincture  or 
osmic  acid,  with  which  they  give  the  same  reactions  as  the  fatty 
oils  but  are  distinguished  from  them  by  their  solubility  in  al- 
cohol. 

XIX.     STEAEOPTENE  (CAMPHOR). 

Literature.  Borscow,  Beitrage  zur  Histochemie  der  Pflanzen 
(Botan.  Zeitg.,  1874,  p.  17jf.)« 

Of  the  group  of  the  stearoptenes,  there  appears  to  have  been 
microscopically  investigated  but  one  body  hitherto,  viz.  : 
Asaron     C^H13O5(?). 

It  is  a  crystallized,  volatile  combination  which  occurs,  princi- 
pally in  the  rhizomes,  in  Asarum  Europeum.  It  is  insoluble  in 
water,  easily  soluble  in  alcohol,  ether,  fatty  and  essential  oils. 
Concentrated  sulphuric  acid  and  fuming  nitric  acid  color  it  orange 
red  or  orange  yellow.  Only  the  first  named  acid  can  be  em- 
ployed in  the  microscopical  investigation.  It  is  thereby  shown 
that  asaron  occurs  in  the  rhizomes,  especially  in  the  peripheral 
layer  of  the  fundamental  parenchyma,  in  cells  which  outwardly 
demonstrate  their  asaron  contents  by  being  filled  with  a  greenish 


442  THE  MICROSCOPE  IN  BOTANY. 

strongly  refractive  substance  (asaron  dissolved  in  an  essential 
oil  not  very  well  known),  To  a  section  lying  in  water  add  a 
drop  of  concentrated  sulphuric  acid  and  it  will  gradually  color 
the  whole  of  the  drops  of  oil  first  yellowish,  then  pure  yellow, 
and  finally  orange.  If  there  are  several  oil  drops  in  the  cell 
they  will,  after  treatment  with  the  acid,  run  together  to  form 
one  or  two  drops  of  larger  dimensions. 


XX.     RESIN,  BALSAM,  TURPENTINE. 

Literature.  Karsten,  Ueber  d.  Entstehung  d.  Harzes, 
Wachses,  etc.  (Botan.  Zeitg.,  1857,  p.  3 13 /".).— Wigand,  Ue- 
ber d.  Desorganisation  d.  Pflzelle,  insbes.  liber  d.  physiol.  Be- 
deut.  v.  Gninmi  u.  Harz  (Pringsheim's  Jahrb.,  Bd.  Ill,  1863, 
p.  115  ff.). — Dippel,  Z.  Histologie  d.  Coniferen  II  (Botan. 
Zeitg.,  1863,  p.  253  jf.).- — Schacht,  Ueber  ein  neues  Secretions- 
organ  im  Wurzelst.  v.  Nephrodium  Filix  mas  (Pringsheim's 
Jahrb.,  Bd.  Ill,  1863,  p.  352/.).— Wiesner,  Ueber  d.  Entsteh. 
d.  Harzes  im  Innern  der  Pflzellen  (Sitzungsber.  d.  K.  Acad. 
d.  Wiss.  Wien,  Bd.  LI,  1865;  see  Chern.  Centralbl.,  1865,  p. 
756/*.).— Muller,  Unters.  liber  d.  Vertheilung  d.  Harze,  etc., 
im  Pflanzenkorper  (Pringsheim's  Jahrb.,  Bd.  V,  1866,  p.  387 
f.)  Hanstein,  Ueber  d.  Organc  d.  Harz-  u.  Schleimabsonderung 
in  den  Laubknospen  (Botan.  Zeitg.,  1868,  p.  697, /*.).—  Fran- 
chimont,  Recherches  sur  Forigine  et  la  constitution  chim.  des 
resines  de  terpenes  (Arch.  Neerland,  t.  VI,  1871,  p.  426,^". 
see  also  Flora  1871,  p.  225 /*.).— Vogl,  Ueber  den  Ban  des 
Holzes  von  Ferreira  spectabilis  u.  d.  Bildungsw.  des  sogen. 
Angelinpedraharzes  (Pringsheim's  Jahrb.,  Bd.  IX,  1873,  p. 

277/.)- 

Principally  in  the  group  of  coniferous  plants,  but  also  in  very 

many  other  growths  even  in  some  cryptogams  themselves  (Ferns, 
Schacht),  are  found  special  conduits  (resin  ducts),  which  are 
produced  by  the  separation  or  absorption  of  the  cells.  In 
these  the  substance  which  we  call  resin  or  balsam  collects ; 
sometimes  it  is  poured  out  as  a  secretion  through  ruptures  or 
cracks.  In  some  cases  it  will  be  conveyed  to  the  outer  world 


RESIN,   BALSAM,  TURPENTINE.  443 

through  peculiar  organs  of  secretion  (conduits  of  leaf  buds, 
resin  in  Hedera). 

Concerning  its  production,  two  views  prevail.  According  to 
one,  which  is  held  by  many  chemists,  resin  is  formed  in  the  plants 
from  essential  oils.  According  to  the  view  of  Wiesner  and 
others,  it  is  produced  from  cellulose  and  starch,  which  are 
transformed  as  a  connecting  link  first  into  tannin  then  into  resin. 
According  to  Franchimont  it  is  formed  from  the  orlycosides. 
These  undergo  a  transformation  into  tannin  and  oxalic  acid  ; 
the  tannic  acid,  which  often  appears  under  the  form  of  glob- 
ules,200 yields,  under  the  influence  of  albuminoid  matter,  a  sub- 
stance (retinogen)  which,  on  its  part,  is  capable  of  producing, 
by  the  influence  of  air,  resin  and  oil  of  turpentine. 

Many  resins  of  the  Coniferce  may  be  designated  by  the  for- 
mula C2oH,0O3  and  may  be  considered  the  oxydation  products 
of  terpene  (for  example  of  the  oil  of  turpentine,  C10H16).  That 
which  we  commonly  name  resin  is  a  solution  of  resin  in  its  nar- 
row sense  in  the  terpenes  (a  mixture  of  carbohydrates,  as  oil 
of  turpentine).  To  the  resins  belong  also  the  balsams.  We 
designate  with  this  latter  name  commonly  those  resins,  which, 
on  account  of  their  larger  turpentine  contents  (about  24  per  cent) 
are  more  fluid,  as,  for  example,  our  balsam  of  fir. 

Resin  appears  in  the  plant  either  as  a  fluid  or  as  a  more  or 
less  solid  granule  (resin-meal,  Wiesner,  as  in  the  pith  cells  and 
wood  parenchyma,  for  example,  in  Acer,  Ulmus,  Facjas,  Quer- 
cus,  Protect,).  Not  infrequently  also  there  occurs  a  mixture  of 
gum  with  resin  which  we  distinguish  as  gum-resin  or  resin-gum 
(see  p.  373). 

Resins  are  seldom  colorless,  mostly  yellow  or  brownish  and 
burn  with  a  sooty  flame.  All  are  insoluble  in  water;  many 
are  soluble  in  alcohol  (giving  a  milky  precipitate  by  the  addi- 
tion of  water)  ;  others  are  insoluble  in  it.  Ether  dissolves 
most  resins  easily,  likewise  sulphuretted  carbon,  oil  of  turpen- 
tine, benzole,  chloroform  and  essential  oils.  (The  resins  of  all 
the  indigenous  Coniferce  are  soluble  in  all  the  dissolving  media 
named.)  Alkalies  and  mineral  acids  also  dissolve  the  resins 
more  or  less  easily. 

800  Qui  pavaissent  devoir  leur  origine  aux  noyaux  cellulaires  ( I  I). 


444  THE  MICROSCOPE  IN  BOTANY. 

At  the  present  time  the  following  methods  are  proposed  for 
the  microscopical  testing  of  resins. 

According  to  Unverdorben  many  resins  form  a  green  combi- 
nation with  copper  salts.  On  this  characteristic,  Franchimont201 
founds  the  following,  experiment  which  is  applicable  to  many 
but  not  to  all  resins.  ' 

Put  the  whole  of  the  part  of  the  plant  containing  resin  for 
several  days  in  an  aqueous  saturated  solution  of  copper  ace- 
tate.202 Wash  it  out  in  water  and  prepare  the  section  from  it. 
The  resin  canals  appear  then  of  a  beautiful  emerald  green  color. 

With  alcanna  tincture  resin  colors  a  beautiful  cinnabar  red. 
The  process  to  be  followed  here  has  already  been  described  on 
page  310.  One  can  use  the  tincture  itself  applying  a  drop  to 
the  preparation  which  is  being  tested.  The  specimen  must  be 
put  into  glycerine  before  the  alcohol  is  fully  evaporated,  be- 
cause otherwise  the  resinous  alcanna  tincture  will  collect  itself 
in  the  form  of  minute  drops  on  the  walls  of  the  cells  and  the 
results  be  impaired. 

Hanstein's  aniline  solution  colors  many  resins  a  beautiful 
blue,  and  it  is  indeed  a  pure  blue  without  violet  or  green.  It 
colors  many  balsams  a  verdigris  green,  also  pure  or  smutty 
olive-green.203 

With  concentrated  sulphuric  acid  the  contents  of  many  resin 
ducts  become  red  or  brown-red.  The  resin  of  many  species  of 
Araucaria  take  a  similar  color  with  sulphuric  acid  or  potassium 
bichromate. 

Resin  meal.  It  is  represented  by  globular  or  flattened  gran- 
ules of  0.0018  to  0.018  mm.  in  diameter,  which  are  homo- 
geneous within  or  contain  a  nucleus  of  a  different  refractive 
power;  one  seldom  remarks  a  lamination  in  them.  They  are 
not  altered  by  distilled  water,  but  by  boiling  some  remain  un- 
changed and  others  are  melted.  Dilute  alcoholic  iodine  does 
not  alter  them,  only  some  take  a  faint,  blue  color.  Boiling 
alcohol  or  ether  dissolves  but  a  few  grains  altogether,  most  of 

201  Franchimont,  1.  c.,  t.  VI,  p.  427,  ff. 

202  According  to  Franchimont  one  may  also  recognize  other  substances  by  means  of  the 
copper  acetate.     Tannin  becomes  brown  therewith  and  glycose  separates  out  metallic 
copper. 

203  Hanstein,  1.  c.,  p.  747. 


EESIN,  BALSAM,  TURPENTINE.  445 

them  only  becoming  clearer  and  stratified.  Potassium  lye  dis- 
solves them  with  saponification  and  brown  coloring,  as  also  does 
ammonia,  though  the  latter  works  less  intensively.  In  cupr- 
ammonia  they  are  unchanged,  likewise  they  are  very  re- 
sistant towards  muriatic  and  nitric  acid.  With  sulphuric  acid 
they  often  become  brown  or  dark  red  ;  with  muriatic  acid,  red  ; 
but  in  nitric  acid  they  bleach  out.  In  dilute  chromic  acid  many 
granules  are  disintegrated  and  most  of  them  become  distinctly 
laminated.  Those  which  have  lain  for  a  long  time  in  chromic 
acid  are  colorless  and  show  the  reaction  of  cellulose  with  iodine 
and  sulphuric  acid,  or  with  cuprammonia.  Ferric  chloride 
gives  with  an  olive  green  or  deep  blue  coloring  the  tannin 
reaction. 

Gammy  resin.  The  study  of  this  substance  is  best  prosecuted 
by  the  help  of  Hanstein's  aniline  mixture.  The  preparation 
treated  with  it  and  carefully  worked  shows  the  resin  beautifully- 
blue,  while  the  gums,  the  amyloids  and  other  muculent  carbohy- 
drates, which  appear  in  the  blue  resin  in  the  form  of  large  or 
small  drops,  either  remain  uncolored  or  take  on  a  pale  rose- 
red  or  strong  reddish  color,  the  specimen  giving  a  beautiful 
image  when  thus  prepared.204 

1.    BETULO-RESIN  ACID. 

Literature.  Muller,  Einige  Bemerk.  liber  d.  harzartigen 
AusscheidungenandenBirken  (Botan.  Zeitg.,  1845,  p.  793).— 
Kosmann  in  Journ.  d.  Pharm.  (2),  Bd.  XXVI,  p.  107. — Mi- 
kosch,  Ueber  d.  Organe  der  Ausscheidung  d.  Betuloretinsaure 
an  derBirke  (Oestcrr.  Bot.  Zeitschr.,  1876,  p.  213,  /I). 

This  resin  acid  (CseHgeOg)  is  insoluble  in  water,  soluble  in 
alcohol,  ether,  alkalies,  carbonic  acid  alkalies  and  concentrated 
sulphuric  acid  (in  the  latter  with  a  red  color).  It  is  secreted 
by  the  hair  glands  on  the  leaves  of  Betula  alba,  and  indeed  the 
secretion  takes  place  by  the  raising  up  of  the  cuticle.  The  con- 
tents of  the  secreting  cells  is  first  a  homogeneous  green  (resting 
on  the  chlorophyll),  but  afterwards  this  color  is  displaced  by  a 
deep  red-brown.  The  secretion  is  a  pale  yellow,  syrupy  mass, 

8«  See  Hanstein,  I.  c.,  Taf.  XI,  Fig.  23. 


446  THE  MICROSCOPE  IN  BOTANY. 

in  which  the  betulo-resin  acid  is  found  dissolved,  in  some  man- 
ner as  yet  unknown.  It  separates  as  a  solid  from  it.  The  contents 
of  the  younger  glands  are  colored  yellow  with  concentrated 
potassium  lye,  afterwards  brick  red  (Mikosch). 


2.    MILK-SAPS. 

Literature.  Weiss  und  Wiesner,  Beitr.  z.  Kenntnisse  d. 
chem.  u.  physik.  Natur  des  Milchs.  d.  Pfl.  (Bot.  Zeitg.,  1862, 
p.  125,  jf.). — Hanstein,  D.  Milchsaftgef.  u.  vervvandten  Or- 
gane  d.  Rinde,  Berlin,  1863. — Dippel,  Beitr.  z.  Histologie  d. 
Pfl.  1.  D.  milchsaftfiihrenden  Zellender  Hollunderarten  (Verh. 
d.  naturhist.  Ver.  d.  pr.  Rheinl.  u.  Westf.,  1865,  Jahrg. 
XXII. — Dippel,  Entstehtmg.  d.  Milchsaftgef.  u.  deren  Stellung 
im  Gefassbiindelsystem;  Rotterd.,  1865. — Vogl,  Ueber  Milch- 
saftgef. d.  Klette  (Botan.  Zeitg.,  1866,  p.  193,  /'.). 

The  milk-saps  represent  a  mixture  of  different  kinds  of 
materials.  It  is  therefore  difficult  to  arrange  these  plant  sub- 
stances in  respect  to  their  chemical  qualities.  But  since  it 
appears  that  resin  always  occurs  in  them,  we  have  concluded 
to  say  a  few  words  concerning  them  in  this  section. 

Weiss  and  Wiesner206  give  as  elements  of  the  milksaps  of 
Euphorbia  platyphylla :  resin,  caoutchouc,  essential  oils,  al- 
buminous matter,  starch,  sugar,  fat,  extractive  matter,  tartaric 
acid,  mineral  elements  and  a  coloring  matter. 

Milk-sap  coagulates  with  a  red  coloring  on  coming  to  the  air. 
With  iodine  solution  it  runs  into  yellow  colored  balls  or 
brown  masses.  Ammonia  turns  it  greenish  (the  develop- 
ment of  the  coloring  matter),  sulphuric  acid  a  beautiful  yellow 
with  coagulation. — "  Put  a  drop  of  sulphuric,  nitric  or  muriatic 
acid  on  a  slide,  and  then  let  a  small  drop  of  the  milk-sap  fall 
upon  it  and  it  will  always  spread  out  into  a  disk-shaped  form 
and  these  little  disks  are  either  beautiful  yellow  (with  sulphuric 
acid)  or  almost  colorless,  only  very  little  yellow  (with  nitric 
acid),  or  again  almost  colorless  with  a  dull  shade  of  yellow-red 
(with  muriatic  acid).  If  one  puts  a  drop  of  the  milk-sap  on  a 

B0«  Weiss  and  Wiesner  in  Bot.  Zeitg.,  1862,  p.  126. 


THE  COLORING  MATTER  OF  FLOWERING  PLANTS.         447 

solution  of  iodine  and  observes  the  disk  thus  produced  with  re- 
flected light,  it  appears  to  the  unaided  eye  a  beautiful  ultra- 
marine blue.  Examination  with  the  microscope  shows  that  this 
blue  does  not  rest  upon  the  amylum  in  the  milk-sap,  since  the 
disk  by  transmitted  light  is  no  longer  blue  but  yellow"  (Weiss 
and  Wiesner,  I.  c.). 


XXI.     THE  COLORIXG  MATTER  OF 
FLOWERING  PLANTS. 

Literature.  Decaisne,  Rech.  anat.  et  phys.  sur  la  Garance 
et  le  developpem.  de  la  matiere  colorante,  Bruxelles,  1837. — 
Nageli  u.  Schwendener  Mikroskop  ,  p.  503,^". — Weiss,  All- 
gem.  Bot.,  Bd.  I,  p.  137. 

The  coloring  substances  to  be  described  here  constitute  the 
color  of  certain  roots,  woods,  seed  scales,  seeds,  etc.  They  have 
been  on  the  whole  but  little  investigated.  It  seems,  however, 
that  at  first  they  appear  on  the  inside  of  the  cell  dissolved  in 
the  cellulose  and  afterwards  passing  into  the  membrane,  be- 
came intercalated  and  permanently  remain.  We  will  here 
describe  some  of  those  which  have  been  better  investigated,  on 

O  ' 

the  authority  of  Xageli  and  Schwendener. 

1 .  Coloring  matter  of  the  Rubia  tinctorum.     Sections  of  the 
fresh  root  appear  yellow  and  not  red  ;  most  of  the  bark  cells  con- 
tain a  yellow  fluid  which  in  the  air  forms  red  flakes.     Young  roots 
possess  altogether  colorless  membranes,  the  pigment  being  found 
alone  in  the  cell  contents.     Potash  colors  it  purple  red,  acids 
orange  color,  ferric  chloride  orange  and  at  last  brownish  red, 
alcohol  extracts  the  yellow  coloring  matter  not  the  altered  red. 

2.  Coloring  matter  of  Colored  Woods.    Well-known  exam- 
ples of  colored  woods  are  the  Brazil  wood,  sandal  wood,  blue 
wood,  Pernambuco  wood,  etc.     The  coloring  matter  permeates 
the  walls,  and  occurs  also  in  the  form  of  small  granules,  in  the 
medullary  rays  and  single  wood  cells. 

In  microscopic  sections  the  coloring  matters  appear  yellow 
to  red  orange.  They  are  soluble  in  alkalies  (with  red,  blue  or 
violet  color,  acids  restore  the  original  color).  They  are  like- 


448  THE  MICROSCOPE  IN  BOTANY. 

wise  dissolved  in  water,  alcohol  and  ether  as  well  as  in  glycerine. 
The  aqueous  solutions  are  mostly  red  or  bluish,  the  alcoholic 
yellow  or  orange.  Acids  dissolve  them  with  yellow,  carmine 
or  blood  red  coloring.  The  aqueous  extract  of  the  red  or  blue 
woods  are  colored  with  ferric  chloride ;  first,  yellow,  then  blue 
(tannin  reaction). 

3.  Coloring  matter  of  the  Barberry-root.      This  pigment  is 
likewise  at  first  dissolved  in  the  cell-sap  and  passes  from  there 
into  the  membrane  which  it  colors  yellow.     Dilute  acids  separ- 
ate out  small  yellow  drops  (albuminous  combinations).     Potash 
causes  a  yellow  precipitate  and  gradually  extracts  from  it  an 
orange  yellow  colored  pigment.     In  a  cold  aqueous  extract  of 
the  coloring  matter  muriatic  acid  produces  radiating  groups  of 
crystal  needles  (berberidin). 

4.  Red  coloring  matter  of  Abrus  precatorius.     The  scarlet 
red  coloring  is  produced  by  a  red  pigment  which  is  intercalated 
in  the  thick  walls  of  the  palisade-like  cells  which  form  the  upper 
surface  of  the  testa.     Alkalies  color  it  blue,  acids  a  high  red. 
It  behaves  also  like  anthocyan  (see  p.  423). 


XXII.  THE  COLORING  MATTER  OF  THE 
CRYPTOGAMIC  PLANTS. 

1.    THE  COLORING  MATTER  OP  ALQ^I. 

Literature.  Ktitzing,  Phycologia  general  is.,  1843,  p.  21. — 
Kiitzing,  Grundz.  d.  philos.  Bot.  Bd.  I,  p.  166. — Colin,  Beitr. 
z.  Physiol.  d.  Phycochromaceen  u.  Flori-deen  (Schultze's  Arch, 
f.  mikrosk.  Amit.,  Bd.  Ill,  1867,  p.  I,/.).— Nageli  u.  Schwen- 
dener,  Mikrosk.,  p.  497,^". — Askenasy,  Beitr.  z.  Kenntii.  d. 
Chlorophylls  u.  einiger  class,  begleit.  FarbstoiFe  (Bot.  Zeitg., 

1867,  p.  225,  ff.).—  Rosanoff,  Observ.  sur  les  fonctions  et  Tes 
proprietes  des  pigments  de  diverses  algues   (Mem.  de  la  soc. 
imp.  de  Cherbourg,  t.  XIII,  1867,  p.  145, /".).—  Kraus  et  Mil- 
lardet,  Etude  s.  la  mat.  color,  des   Phycochromacees    et   dcs 
Diatomees  (Mem.  de  la  soc.  des  sc.  nat.  de  Strasbourg,  t.  VI, 

1868,  p.  23,  ff.).—  Millardet,  Stir  la  nature    du  pigment  des 


COLORING  MATTER  OF  CRYPTOGAMIC  PLANTS.     449 

Fucoidees  (Comptes  rendus,  t.  LXVIII,  1869,  p.  462,  f.).— 
Kraus,  Z.  Kenntn.  d.   Chlorophyllfarbstoffe,  Stuttg.,  1872.- 
Pringsheim,  Ueber  iiattirl.  Chlorophyll  modi  f.  u.  d.  FarbstofFe, 
d.  Florideen  (Monatsber.  d.  K.  Acad.  d.  Wiss.  Berlin,  1875, 
p.  749,^".). — Sorby,  On  the  characteristic  coloring-matter  of 
the  red  groups  of  algae  (Journal  of  the  Linn.  Soc.  Vol.  XV, 
1875,  p.  34,^.). —  Reiuke,  Beitrag.  z.  Kenntn.  d.  Phycoxan- 
thins  (Pringsheim's  Jahrb.,  Bd.  X,  1876,  p.  399, jf.).— Nebel- 
ung,   Spectrosk.  Unters.    Uber   d.  Farbst.    einiger   Siisswass- 
eralgen  (Bot.  Zeitg.,  1878,  p.  369./1). 

The  coloring  substances  of  algre  are  all,  so  far  as  they  occur 
as  cell  contents,  joined  with  the  plasma,  very  like  chlorophyll, 
and  are  to  be  included  among  its  modifications.  That  indeed  a 
great  number  of  the  algse  possess  true  chlorophyll  is  a  well 
known  fact ;  while  in  others,  on  the  other  hand,  a  green  coloring 
matter  occurs  which  is  distinct  from  true  chlorophyll  in  its 
chemical  as  well  as  in  its  spectroscopic  behavior.  The  coloring 
substances  of  algre  may  be  divided  into  two- groups  in  respect 
to  their  behavior  towards  alcohol  and  water,  those  of  the  one 
being  soluble  in  alcohol  but  in  water  insoluble,  and  those  of  the 
other  soluble  in  water,  but  mostly  not  in  alcohol. 

The  more  exactly  investigated  are  the  following. 

A  B 

Soluble  in  alcohol,  not  in  water.  Soluble  in  water,  not  in  alcohol. 

1.  Floriclia  green    (green).  4.   Pliykoer>thrin    (red;. 

5.    Phykoxanthiu    (yellow).  5.   Phykocyau    (blue). 

3.   Diatomiii   (yellow-brown).  6.   Palmellin   (red). 

7.   Phykophaein   (brown). 

1.  Floridia  green  is  extracted  from  the  Floridia  by  means  of 
alcohol,  represents  a  variety  of  chlorophyll,  and  is  really  very 
like  it. 

2.  Phykoxanthin.     Yellow  coloring  matter  in  kelp  and  nu- 
merous fresh  water  algae  in  connection  with  protoplasmic  bodies. 
It  is  easily  extracted  from  the  former  by  means  of  40  per  cent 
alcohol  which  will  not  dissolve  true  chlorophyll.     Evaporate  the 
solution  and  the  coloring  matter  remains  behind  a  slimy,  amor- 
phous, brown  mass.      Alcoholic  solutions  of  phykoxanthin  are 
made  blue  green  by  acids  but  are  not  changed  by  alkalies. 

29 


450  THE  MICROSCOPE  IN  BOTANY. 

3.  .Diatomin  (endochrom).     The   yellow   to   brown-yellow 
coloring  matter  of  the  Diatomacece.     It  becomes  greenish  by 
application  of  acids  and  alkalies  and  with  concentrated  sulphuric 
acid  a  beautiful  verdigris  geeen.     It  consists  of  phykoxanthiu 
and  chlorophyll. 

4.  Phykoerythrin  (Floridia  red).     The  red  coloring  matter 
of  the  Floridia  appears  the  same  when  dry,  is  soluble  in  water 
but  not  in  alcohol  or  ether.     The  aqueous  solution  loses  its  color 
in  the  light.     Alkalies  color  it  pale  olive  green  (almost  color- 
less) ,  acids  restore  the  red  color  again.     Concentrated  sulphuric 
acid  does  not  alter  the  aqueous  extract. 

5.  Phykocyan   (Phykochrom).     In    blue-green    alga3.     The 
blue-green  or  indigo  blue  coloring  matter  is  soluble  in  water 
but  not  in  alcohol,  becomes  yellowish  brownish  or  yellow  green 
with  alkalies  ;  with  muriatic  acid,  orange  red  or  smutty  orange. 

6.  Palmellin.    A  red  coloring  matter  of  PorpJiyridium  which 
is  soluble  in  water  and  becomes  blue  with  alkalies. 

7.  Phykophaem.     A  brown  coloring  matter  from  the  Fuca- 
cece,  soluble  in  water  and  dilute  alcohol,  but  insoluble  in  absolute 
alcohol,  ether  and  benzole.     It  is  intercalated  in  the  protoplas- 
mic grains  in  connection  with  chlorophyll   and  phykoxanthin. 
The  aqueous  solution  is  intense  brown  red  but  is  not  fluorescent. 
Absolute  alcohol,  cold,  produces  a  cloudiness  in  the  solution. 
By  warming,  the  coloring  matter  is  thrown  down  as  a  partly 
flaky  brown  precipitate.     A  like  effect  is  produced  by  muriatic 
nitric  and  sulphuric  acids.     Concentrated  alkalies   bleach  the 
solution  somewhat. 

The  above  described  coloring  substances  commonly  occur  in 
connection  with  chlorophyll  and  its  optical  effect  mixes,  in  the 
living  plant,  with  that  of  chlorophyll. 

Spectroscopic  behavior.  Some  of  the  above  named  coloring 
substances  of  the  algre  have  been  subjected  to  critical  spectro- 
scopical  studies,  while  others  are  in  this  regard  quite  unknown. 
The  following  have  been  exactly  tested  spectroscopically. 

Floridia-green  and  Floriaia-red  (Pringsheim).206  The  Phy- 
koerythrin (Florida-red)  shows  a  spectrum  which  possesses  all 

aoe  Pringsheim  in  Monatsber.  d.  K.  Acad.,  Berlin,  1875,  pp.  749-751. 


COLORING  MATTER  OF  CRYPTOGAMIC  PLANTS. 


451 


the  essential  marks  of  the  chlorophyll  spectrum  (Fig.  150, 
constructed  after  Pringsheim's  method  I.  c.,  with  Angstroms 
scale  for  a  standard  ;  0,  10,  20,  etc.,  gives  the  optical  concentra- 
tion of  the  solution  under  investigation ;  the  dotted  line  is  the 
absorption  curve  of  the  phykoerythrin,  the  full  line  that  of  the 
Floridia-green).  But  the  chlorophyll  bands  III,  IV  and  IV« 
(see  p.  413,  ff.)  appear  to  be  considerably  strengthened  in  the 
phykoerythrin,  bands  I  and  II  being  very  much  weakened 
while  the  bauds  in  the  blue  and  violet  remain  unchanged  in 
their  intensity.  With  some  coloring  substances  which  belong 


B  C 


so 


FIG. iso. 


to  the  phykoerythrin  group,  there  appear  to  be  some  minor 
differences  in  the  weakening  of  bands  I  and  II.  But  on  the 
whole  the  maxima  and  minima  of  the  absorption  curve  coincide 
with  those  of  the  absorption  curve  of  chlorophyll. 

The  green  alcoholic  extract  of  the  Floridia  (Fig.  150)  is 
spectroscopically  somewhat  different  from  chlorophyll.  Its 
spectrum  differs  from  that  of  chlorophyll  by  a  slight  weakening 
of  bands  I,  II  and  III,  and  by  a  considerable  strengthening  of 


452 


THE  MICROSCOPE  IN  BOTANY. 


band  IV,  and  of  the  bands  in  the  blue  and  violet,  which  in  a 
medium  optical  concentration  flow  together  to  make  an  end 
absorption,  and  finally  by  a  new  maximum  of  absorption  which 
includes  the  wave  lengths  51  and  49  (unit—  0.00001  mm.). 

By  comparing  the  spectra  with  each  other  (Fig.  150)  there 
is  seen  to  be  a  very  exact  coincidence  of  the  absorption  maxima 
and  minima,  from  which  it  becomes  apparent  that  Floridia-red 
is  a  modification  of  Floridia-green,  and  not  a  direct  modification 
of  the  phanerogamic  chlorophyll  (Pringsheim). 


75    70      C3 


i     !  r'l          1                 '       I 

.    i  !,i  '  I'  •  ••  '  ' 

1 

aB  C 


E.I) 


G 


H 


FIG. 


Phykocyan  (Reinke).  The  clear  blue,  aqueous  extract  of 
Oscillaria,  which  has  a  red  fluorescence,  gives  in  a  layer  15  cm* 
thick  a  spectrum  with  four  absorption  bands  (Fig.  151  after 
Reiuke),  of  which  III  is  very  weak*  If  the  solution  be  boiled 
only  the  bands  at  F  and  H  remain  visible,  likewise  it  loses  its 
fluorescence. 

If  after  the  extraction  of  the  phykocyan,  the  Oscillaria  be 
again  subjected  to  extraction  by  alcohol,  and  the  filtrate  shaken 


75    70 


I 

i 

1       !       1    .--'•'.-'  •'    1 

':    '           1 

,  I  i  I  U  j 

-;.,! 

aB  0 


JD 


Eb 


FIG.  152. 


H 


up  with  benzole,  phykoxanthin  is  retained  in  the  alcohol  as  an 
amber  yellow  fluid.  This  gives  a  spectrum  like  that  of  chloro- 
phyll (Fig.  152  after  Reinke),  with  this  difference  that  band  II 
shows  a  not  unimportant  broadening  towards  the  red  end  of  the 
spectrum.  It  even  divides  sometimes  into  two  bands  ;  band  III 
is  also  broadened  towards  the  red  side.  Of  the  bands  of  the 


COLORING  MATTER  OF  CRYPTOGAMIC  PLANTS.  453 

second  half  of  the  spectrum,  VI  and  VII  coincide  with  the 
corresponding  chlorophyll  bands,  IV  and  V  differ  from  them207. 

Concerning  the  older  spectroscopical  investigations  of  the 
coloring  matter  of  algae,  which  were  commonly  made  without 
specifying  the  optical  concentration  and  have  therefore  only 
relative  value,  one  may  compare  the  above  cited  writings  of 
Cohn,  Kraus,  Millardet,  Rosanoff,  and  Askenasy. 

It  may  be  remarked  by  way  of  appendix  that  Nageli208  mentions 
two  membrane  coloring  substances  of  algae,  viz.,  Gloeocapsin 
and  Seytonemin,  Gloeocapsin  occurs  in  the  membranes  of  Gloeo- 
capsa  and  some  other  algae,  and  is  a  red  or  blue  coloring  mat- 
ter which  is  colored  rose,  red  orange,  or  brown  red  by  muriatic 
acid,  and  with  potash  lye  blue  or  blue-violet.  Sc}^tonemin  is  u 
yellow  or  dark  brown  coloring  matter  in  the  walls  of  the  Phy- 
cromacecB  which  becomes  verdigris  green  with  muriatic  acid,  and 
with  alkalies  yellow,  often  almost  gold  yellow. 

2.    FUNGI  CoLOKiNQ  MATTER. 

Literature.  Schroter,  Ueber  eininge  durch  Bacterien  ge- 
bildete  Fermente  (Conn's  Beitrag.  z.,Biol.  d.  Pfl.,  Bd.  I,  1872, 
p.  109,  ff.). — Klein  in  Quart.  Journ.  of  Microsc.  sc.,  1875,  p. 
381,^. — Sadebeck,  Durch  mikrosk.  Organismen  rothgef.  TTnss- 
er  (Verf.  d.  bot  Ver.  d.  Prov.  Brandenburg,  Bd.  XVII,  1876, 
p.  77,/.) — Cugini,  Sulla  materia  colorante  del  Boletus  luridus 
(Gazetta  chimicu,  Vol.  VII,  1877,  p.  209,/".). 

In  the  group  of  fungi  coloring  substances  of  very  different 
nature  seem  to  occur  in  great  numbers  all  of  which  have  nothing 
whatever  to  do  with  chlorophyll.  Alas  !  that  these  substances 
have  not  been  at  all  studied.  We  can  therefore  give  here  only 
some  altogether  superficial  statements. 

First.  Coloring  matter  frequently  occurs  in  the  Schizomyce- 
tece.  The  color  producing  bacteria  show  different  colored  pig- 
ments (red,  yellow,  green,  blue,  brown)  of  intense  shades. 
They  are  insoluble  in  water,  alcohol  and  ether.209  Alcohol  and 

207  Reinke  in  Pringsheim's  Jahrb.,  Bd.  X,  p.  406,  ff. 
209  Nageli  u.  Schwendener,  Mikr.,  p.  507. 

209  According  to  Sadebeck,  1.  c.,  the  red  pigment  of  micrococcus  is  partly  soluble  in 
water. 


454  THE  MICROSCOPE  IN  BOTANY. 

ether  remove  the  red  from  the  bacteria  pigment,  potash  solu- 
tion makes  it  transparent.  The  red  coloring  matter  of  Micro- 
coccus  is  according  to  Helm210  aniline  red.  With  muriatic  acid 
it  becomes  rose  colored,  likewise  with  sulphuric  acid  (violet 
with  the  addition  of  more  acid),  with  alkalies  yellow.  In  this 
case  acids  restore  the  red  color. 

The  coloring  matter  of  Agaricus  atrotomentosus  dissolves  in 
alcohol  and  acetic  acid  with  rose  red  color,  becomes  yellow  with 
alkalies  and  is  insoluble  in  water  and  benzole. 

According  to  Phipson  aniline-like  coloring  matters  occur  like- 
wise in  Boletus  luridus  and  B.  cyanescens.  Cugini  controverts 
this  and  gives  the  following  characteristics  of  the  "acid-like" 
coloring  matter  of  Boletus  luridus.  It  is  soluble  in  water  and 
alcohol,  acids  color  it  a  beautiful  yellow  (chromic  acid,  yellow 
brown),  ammonia  blue,  potash-lye  red.  Ferric  chloride  gives  it 
an  intense  green  color. 

Spectroscopically  the  fungi  coloring  substances,  with  the 
exception  of  the  bacteria  pigment,  have  not  been  investigated. 
The  spectra  have  not  the  remotest  likeness  to  those  of  chloro- 
phyll coloring  matter. 

«i°  O.  Helm  in  Avch.  f.  Pharm.,  1875,  p.  19,  #. 


INDEX. 


Abbe  condenser,  89. 

Abbe's  binocular  ocular,  41. 

Aberration,  chromatic,  correcting,  28. 
"       "       spherical  and  chromatic,  5. 
"       «  "  "      correcting,  26, 

27. 

Aberration,  spherical,  illustrated,  25. 

Abrus  pectorus,  red  coloring  matter  of, 
448. 

Absorption  spectra,  153. 

'•  "       comparison  of,  154. 

•'  "       measuring,  154. 

"        of  chlorophyll,  415. 
"       represented  by  curves,  420. 

Accessories,  microscopical,  100. 

Acetic  acid,  297. 

"       "       to  prepare  a  1  per  cent  solu- 
tion of.  282. 

Acetic  acid  carmine  for  cell  nucleus,  400. 

Achromatic  lenses,  set  of.  23. 

"          microscope  first  made  by  Van 
Deyl,  6. 

Achromatic  triplet,  101. 

Adjustment,  the  fine,  77. 

Agaricus  atrotomentosus,  coloring  matter 
of,  454. 

Aids  to  microscopical  drawing,  254. 

Air  pump,  a  home  made,  197. 

Alantin,  375. 

Albuminous  matter,  379. 

Alcanna  tincture,  310. 

'•        and  proteid  matter,  385. 

Alcohol.  297. 

Alcoholic  solution  of  chlorophyll,  411. 

"         and  nitric  acid  as  a  bleaching 
medium,  202. 

Aleuron,  380. 

Algae,  coloring  matter  of,  448-9. 
"        and  fungi  examined  without  pre- 
paration, 161. 

Alkalies  and  cellulose,  324. 

Alkaloids,  438. 

Alum  carmine  and  cellulose,  325. 

Amber  cement,  King's,  235. 

Amici's  aplanatic  lens,  6. 

Ammonia,  291. 

Amorphic  proteid  grains,  382. 

Amyloid  mucilage,  368,  370. 


Angle  of  aperture.  24. 

"  "       large  in  lens  systems, 

27. 
Aniline  coloring  matter,  299. 

"       colors  for  cell  nucleus,  401. 
"       fuchsin  solution,  300. 
"       green  and  violet,  301. 
"       method  of  staining  with,  300. 
"       mixture,  Hanstein's,  299. 
*'       sulphate,  301. 
"  "       and  lign'm,  233. 

"  "       reaction    on    epidermal 

tissue,  353T 
Anthocyan,  422-3. 
Anthoxanthin,  413,  422,  423. 

"  spectrum  of,  423,  424. 

Aplanatic  lenses,  6,  28. 
Apparatus   for    the   preparation   of  "re- 
agents, 272. 
Arabin,  371,  374. 
Asaron,  441. 

Asbestos  for  filtering,  273. 
Asparagin,  298,  425. 
"  crystals,  427. 

"  "       testing,  428. 

"  method  of  investigating,  426-7. 

physiological  function  of,  426. 
Asphalt  varnish  for  cement,  234. 


B. 


Baber's  picro-carmine,  309. 
Balsam.  442. 

Canada,  222. 
Bands  of  chlorophyll  absorption  spectra, 

415.  416. 

Band  I,  characteristic  of  chlorophyll,  418. 
Barberry  root,  coloring  matter  of,  448. 
Bark  of  Lonicera  stained  bluish,  269. 
Bassorin,  371,  374. 

Bast  of  Lonicera  colored  reddish,  269. 
£atracheospermummoniliforme,mounting. 

233. 
Beale's  carmine  solution,  307. 

"  "       for  cell  nucleus,  400. 

"  "        for  spirogyra,  308. 

Behren's  cuprammonia,  294. 
Bell-glasses,  175. 
Benzole  chlorophyll,  412. 

(455) 


456 


INDEX. 


Berthollctia  cxcelsa,    crystalloids    in,  386, 

387. 

Bessey's  laboratoiy  table,  253. 
Betula-resin  acid,  445. 
Binocular  micro-spectroscope,  143. 
"         ocular,  38. 
"  "    Abbe's,  41. 

"  "    Nachet's,  39,  41. 

"    Stephenson's,  40. 
"  "    Tolles',  39. 

"  "    Wenham,  40,  41. 

Bleaching  with  alcohol  and  nitric  acid,  202. 
"       bacteria  with  carbolic  acid,  201, 
202.      - 
Bleaching  with  calcium  chloride,  202. 

with  chloride  of  lime  and  car- 
bonate of  soda,  200. 
Bleaching  with  chlorine  gas,  201. 

"         cork  cell   walls  with   chromic 
acid,  202. 
Bleaching  with  potassium  alcohol,  200. 

"  "  "       hydroxide,  199, 

200. 
Bluing  cellulose,  321,  322. 

"       starch  with  iodine,  360. 
Blue  vitriol,  293. 

Boletus  luridus,  coloring  matter  of,  454. 
Borel's  account  of  Janssen,  3. 
Botanical   dissecting   microscope,  Zent- 

mayer,  107. 

Bb'ttoher's  cuprammonia,  294. 
Bottle  for  glycerine,  21!). 
Box  for  slides,  248. 
Brewster's  achromatic  globe,  6. 
Brewster's  measuring  apparatus  for  mi- 
cro-spectroscope, 145-148. 
Browning's  micro-spectroscopic  measur- 
ing apparatus,  use  of,  148, 149. 
Brown's  rubber  cement,  238. 
Brack's  compound  dissecting  microscope, 

107. 

Bulb-burette,  275. 
Bull's-eye  condensers,  93. 
Bunsen  gas  burner.  273. 
Burette,  the  bulb,  275. 

"       Mohr's  spring  compressor,  277. 
"       use  of,  277. 
Butterflies' scales  as  test  objects,  57,  58, 

59,  60,  61. 

Butter  plates  used  for  porcelain  dishes, 
173. 


c. 

Cabinets  for  slides,  249. 
Calcining  plants,  164. 
Calcium  carbonate,  431. 

"          incrustations,  to    re- 
move, 164. 


Calcium  chlorate  dryer,  175. 

"          swells  starch,  and  chlo- 
rophyll grains,  158. 
Calcium  chloride  for  bleaching,  202. 
"       oxalate,  431. 
"        salts,  430. 
"       sulphate,  431. 

Caldwell's  automatic  microtome,  N.,  195. 
Cambium  zone  of  Lonicera,  269. 
Camera  lucida,  drawing  with,  258. 
"       Grunow's,  118. 
"       as    measuring     appara- 
tus, 127. 
Camera  lucida,  Nobert's,  117. 

"  "       the  Wollaston,  115,  116. 

Camphor,  441. 
Cane  sugar,  299,  377. 
Canada  balsam  for  mounting,  222. 

"       method  of  using,  222. 
Carbo-hydrates,  314. 
Carbolic  acid,  302. 

"    for  bleaching  bacteria,  201. 
"          "    for  preserving  plant  tissue, 
227. 

Card  catalogue  for  preparations,  246-7. 
Carmine  and  protoplasm,  207. 
"       solutions,  306. 

"       stains    for    protoplasmic     sub- 
stances, 309. 
Castlehun's  preserving  fluid  for  lichens, 

227. 

Cataloguing  preparations,  264. 
Caustic  potash,  288. 
Cell  nucleus,  397. 

"    fixing,  398. 
"  "    staining,  399. 

"    contents,  table  of  reactions  of,  403. 
Cellulose,  313. 

"    and  alkalies,  324. 

"      "    alum  carmine,  325. 

"    cork,  318,  347. 

"    and  cuprammonia,  324. 

"    dissolved  in  sulphuric  and  chromic 

acid,  323. 
"    essential,  317. 
"    fungus,  318,  353. 
"    and  iodine  reagents,  319,  323. 
"        "    mineral  acids,  323. 
"        "    its  modifications,  315. 
"    mucilaginous,  327. 
"    muculent,  317. 
"    in  the  narrow  sense,  318. 
"    and  potassium   copper   sulphate, 
325,  326. 

Cellulose  reactions,  table  of,  356. 
"    reagents  for,  318. 
"    and  the  sulphuric  acid  and  iodine 
reaction,  323, 324. 
Cellulose,  wood,  317. 


INDEX. 


457 


Cement,  asphalt  varnish.  234. 
"    Brown's  rubber,  238. 
"    cells,  method  of  making,  231-2. 
"    copal  varnish,  236. 
"    dammar  varnish,  236. 
"    gold  size,  236. 
"    King's  acquer  finish,     6. 
11       "       white,  236. 
"    mastic  varnish,  234. 
"    shellac  and  sealing  wax,  234. 
"    wax,  234. 
"    white  zinc,  238. 

Cementing  angular  cover-glasses,  237. 
"       cells  by  heat,  244. 
"       and  finishing  the  monnt,  234. 
"       glycerine  mounts,  238. 
Cerasin,  374. 
Chemical  reactions,  268. 
Cherry  gum,  372. 

Cherry  wood  contains  phloroglucin,  303. 
Chevalier's  aplanatic  lens,  6. 
Chloride  of  calcium  for  mounting,  223. 

"       "    lime  and  carbonate  of  soda 
for  bleaching,  200. 
Chlor-iodide  of  zinc,  286,  287. 

"    "    swells  cell  walls,  158. 
Chlorophyll,  403. 

"       absorbent  of  solar  energy,  421. 
"       coloring  matter,  410. 
"       decomposed,     changes     spec- 
trum, 417. 
Chlorophyll  grains,  drawing,  263. 

"  "    fundamental  substance 

of,  408. 
Chlorophyll  grains  and  starch,  364. 

"  "       under    the     spectro- 

scope. 413. 
Chromatic  aberration,  5. 

"  "    correction  of,  28. 

Chromic  acid,  292. 

"          "  for  bleaching  cork  cell  walls, 
202. 

Chromic  acid,  testing  solution  of,  282. 
Chromogen,  tannin  a,  436. 
Chrysophanic  acid,  4:>5. 
Circular  cover-glass,  mounting  with,  239. 
Clarification  of  the  preparation,  198. 
Classification  of  plant  substances,  312, 313. 
Cleaning  the  lenses,  97. 

"       slides  and  cover-glasses,  216. 
Cobweb  micrometer,  127. 
Cochineal  extract,  305. 
Cocldington  lens,  the,  101. 
Colored   granules  in  flowers  and   fruit, 

424,  425. 
Colored  mounting  fluid,  226. 

"        wood,  447. 

Coloring  matter  of  cryptogamic  plants, 
448. 


Coloring  matter  of  flowers,  421. 

"  "       •"    flowering  plants,  447. 

Comparison  prism  of  micro-spectroscope, 

141. 
Comparison  table  of  English  and  metric 

scales,  opposite  p.  133. 
Compound  dissecting  microscope,  107,103, 
Compressor,  parallel,  176. 
Condenser,  87. 

the  Abbe,  89. 
bull's  eye,  93. 
"       described,  88. 
"        stops,  90. 
"       the  Webster,  89. 
Conducting  microscopical  drawing,  259. 
Congress  nose-piece,  75. 
Coniferin,  433. 

Coniferous  wood  section,  55. 
Copal  varnish,  a  cement,  236. 
Copper  sulphate,  293.  J^rv+JUU)**.    y  7  O 
Cork  cellulose,  318.  347. 
"    cell  walls,  bleaching,  202. 
"    layer,  chemical  nature  of,  349. 
"    in  section  cutting,  184. 
"    reactions  of,  350,  352. 
Correction  system,  31. 
Corrosive  sublimate,  295. 

"  "  for  mounting,  224. 

Cover-glass,  the,  215. 

"          correction  for,  33. 
"          damage  to  image  by,  32. 
"          putting  on  the,  230. 
"  supports  for,  231. 

"          cementing  angular,  237. 
"  mounting  with  circular,  239. 

Creosote  mixture  for  mounting,  224. 
Cross  thi-eads  for  drawing,  256. 
Cryptogamic  plants,  coloring  matter  of, 

448. 
Crystals,  asparagin,427. 

"       of  calcium  carbonate,  431. 
"       "          "    oxalate,  431. 
"       drawing,  264. 
"       in  proteid  grains,  391. 
Crystalloids  of  Bertholletia,  386,  387. 
"       in  Floridia,  389. 
"       in  Pilobultts,  388. 
"       in  proteid  grains,  385. 
"        Solanum  Americanum,  390. 
"       without  inclosing  mass,  387. 
Culpeper  &  Scarlet  invent  the  mirror,  4. 
Cupramrnonip,  293. 

"          and  cellulose,  324. 
Cupric  acetate,  298. 
Cutting  out  fossil  specimens,  207. 

D. 

Dahlin,  375. 
Dammar  and  mastic  for  mounting,  223. 


458 


INDEX. 


Dammar  varnish  cement,  236. 
Definition,  testing  the,  54. 
Dextrine,  365. 

Diaphragm,  cylindrical,  85. 
"  the  iris,  86. 

"  position  of,  84. 

"  the  revolving,  84. 

"          stops,  special,  86. 
"  "        Ward's,  87. 

Diatomin,  450. 

Diatoms  in  Liverpool  coal,  204. 
"       method  of  cleaning,  430. 
"       mounting  for  test  objects,  62. 
"       as  test  objects,  61. 
"        "    «       "        Fritsch  &  Miiller, 
N.,  64. 
Dissecting  microscope,  compact,  106. 

"  "          handy,  103. 

Distilling  reagents,  273. 
Distribution   of  energy   in   spectrum  of 

chlorophyll,  420. 
Divini's  ocular,  4. 
Dolland's  achromatic  telescope,  5. 
Double  staining,  305. 
Doublets  as  objectives,  5. 
Drawing  apparatus  for  the  microscope, 

110. 

Drawing  with  camera  lucida,  258. 
"       chlorophyll  grains,  263. 
"        conducting   microscopical,  259, 

260. 

"       controlling  the  light  in,  N.,  116. 
"       with  cross-threads,  256. 
"       crystals,  264. 
"       fluid  drops,  264. 

granular  masses,  262. 
With  India  ink,  262. 
materials,  265. 
microscopic  objects,  11,  254. 
by  ocular  micrometer,  254,  255. 
protoplasm,  262. 
"       section  of  Pteris  aquilina,  257. 
"        spiral  tissue,  262. 
"       starch  grains,  263. 
"       wood  cells,  261. 
Drawings  coloring  microscopical,  265. 
Draw  tube,  71. 

"         "     use  of,  72,  73. 
Drebbel,  supposed  inventor  of  the  micros- 
cope, 3. 

E. 

Elder-pith,  embedding  and  cutting  sec- 
tions in,  183-5. 
Embedding  in  glycerine  jelly,  186. 

"        .  "    gum  and  glycerine,  186. 
"          media,  186-7. 
"          for  Providence  microtome, 
190. 


Embedding  in  tallow  and  paraffine,  187. 
Emery  plate  for  grinding  sections,  208. 
Engravers'  glass,  10-2. 
Eosin,  304. 

Epidermis,  to  examine  without  prepara- 
tion, 160. 
Epi plasm,  396. 
Essential  oils,  441. 
Ether,  297. 
Etiolin,  413. 

"    spectrum'of,  419. 
Euler's  achromatics,  5. 
Examples  of  volumetric  method,  281-3. 
Exponent  of  refraction,  158. 
Eye  shade,  Ward's,  43. 


Faber's  pencils  for  drawing,  265. 
Facility  nose-piece,  75. 
Farrant's  medium,  N.,  186. 
Fasoldt's  nose-piece,  76. 
Fats,  439. 
Fatty  oils,  440. 
Fehl ing's  solution,  378. 
Ferric  chloride,  292. 
Ferrous  salts,  tannin  test,  268. 
Field  of  vision,  requirements  of,  43. 
Filter  of  asbestos,  273. 
"     "  glass  wool,  273. 
"    preparing  a,  273. 
Fine  adjustment,  77. 

"  "  Bausch  &  Lomb,  79. 

"  "  Bulloch,  78. 

"  "  Zentmayer,  78. 

Finishing  mounts  on  turn-table,  243,  244. 
Fixing  the  cell  nucleus,  398. 
Flask  for  measuring,  274. 
Flax,  mucilage  of,  c74. 
Flea  glasses,  2. 
Floridia,  crystalloids  in,  389. 
"       green,  449. 
"          "       spectrum  of,  451. 
"       red, 450. 

Flowers,  coloring  matter  of,  421. 
Flowering  plants,  coloring  matter  of,  447. 
Fluid  drops,  drawing,  264. 
Fluids,  table  of  specific  gravity  of,  278. 
Focussing,  coarse  and  fine,  7. 
"          the  objective,  98. 
Fontana,  supposed  inventor  of  compound 

microscope,  3. 
Forceps  and  scissors,  172. 
Fossil  plants,  preparation  of,  203 
Fossil  wood,  grinding  thin,  209. 
Frangulin,  434. 
Fraunhofer,  achromatic  lenses 

"  lines  in  spectrum,  142 

Free  hand  section  cutting,  179. 
Freezing  microtome,  Taylor's,  191. 


INDEX. 


459 


Freezing  mixture,  Hartig's,  163. 
Freezing  mixtures,  other,  192. 

"       seeds  to  separate  cells,  163. 
Fremy's  classification  of  plant  tissue,  N., 

316. 

Frey's  fuchsin  solution,  300. 
"    glycerine  carmine,  306. 
Fritsch  and  Miiller's  diatom  tests,  N.,  64. 
Fuchsin,  Frey's,  300. 
Fundamental    substance  of  chlorophyll 

grains,  408. 
Fundamental  tissue  and  aniline  sulphate, 

335. 

Fundamental  tissue  and  indol,  340. 
Fungi,  coloring  matter,  453. 
Fungus  cellulose,  318,  353. 

•'  "       theories  of,  353-4. 

Fuss'  achromatic  microscopes,  5. 

G. 

Galilei,  supposed  inventor  of  compound 

microscope,  3. 
Gas  slide.  Hunt's,  -252. 
Gerlach's  ammonium  carminate,  306. 
Glass  cells,  making,  222,  233, 
"    rods,  173. 
"    wool  for  filters,  273. 
Globoids  in  proteid  grains.  391. 
Glycerine,  bottle  for  holding,  219. 
"  a  clarifying  medium,  198. 

''  a  mounting  fluid.  218. 

"          jelly,  Kaiser's.  220. 
preparing,  221. 
Norstedt's,  220. 
embedding  medium,  186. 
for  mounting,  220. 
an  aid  in  glycerine  mounts, 
229. 

Glycose,  377. 
Glycoside,  432. 
Gold-size,  cement,  236. 
Goniometer,  133, 137, 138. 
Gb'ppert's,  studies  of  fossil  wood,  204. 
Grammatophora  marina,  67,  68. 

"  oceanica,  68. 

Graphical   representation  of  absorption, 

420. 
Grape  sugar,  377. 

"         <;       testing,  377. 
Grunacher's  alum-carmine,  308. 
Grinding  down  rock  specimens,  208. 

"       preparation  thin,  209. 
Ground  sections  first  made  by  Sorby,  206. 
Growing  slide,  253. 
Grunow's  camera  lucida,  118. 
Gum,  371. 

"      arabic  and  glycerine  for  embedding 
186. 
Gum,  cherry,  372,  374. 


Glim  mastic  and  dammar  for  mounting, 

223. 
Gum  mucilage,  368,  371. 

"      tragacanth,  372,  373. 
Gummy  resin,  445. 

H. 

Haematoxylin,  304. 

"          "     for  cell  nucleus,  400. 
"     double  staining,  305. 
Hair  pencils,  173. 

"  "    for  cementing,  237. 

Hairs  of  plants,  to  examine,  160. 
Hanaman's,  C.  E.,  method  of  cleaning 

slides  and  cover  glasses,  217. 
Handy  dissecting  microscope,  103. 
Hand  rests,  Ward's,  108, 109. 
Hanging  drop,  251,  253. 
Hanstein's  aniline  mixture,  299. 

"  method  of  clarifying  sections 

199. 
Hardening  material  with  alcohol,  178. 

"       with  chromic  acid  and  potassi- 
um bichromate,  178. 
Hardening  with  perosmic  acid,  178. 

"       tissue  by  freezing,  advantages 

of,  194. 
Hartig,  colors  protoplasm  with  carmine, 

267. 
Hartig,  founder  of  microscopical  analysis, 

270. 
Hartig's  ammoniacal  carmine,  306. 

"       freezing  maceration  process,  163- 
"       mixture,  163. 
"       maceration  by  boiling,  163. 
Hartnack,  inventor  of  immersion  lenses, 

30. 

Helenin,  375. 
Hervey's  turn-table,  241. 
Hesperidin,  434. 

Hipparchia  janira,  scale  of,  58,  60,  61,  69. 
Histological      dissecting       microscope, 

Beck's,  108. 
Honing  the  razor,  169. 
Hooke,  Robert,  4. 
How  to  judge  a  microscope,  53. 
Hydriodic  acid,  how  formed,  287. 
Hydrocharis  Morsus-rance,  circulation  in 

root  hairs,  160. 
Hydrochloric  acid,  284. 
Hunt's  gas  slide,  252. 


I. 


Illuminating  apparatus,  82. 

•'  combinations,  90. 

Illuminator,  Beck's,  94. 

"          opaque,  93. 

"          Ward's  iris,  91,  92. 


460 


INDEX. 


Immersion  system,  30. 
Incinerating  plants,  164. 
India  ink,  drawing  with,  262,  265,  266. 
Indol,  303. 

"      and  lignin,  338. 
Inorganic  matter  in  proteid  grains,  390. 

"          plant  substances,  428. 
Intercalation  in  cellulose  walls,  316. 
Intercellular  substance,  317,  343. 

«  "         the  theory  of,  344. 

Instruments  for  making  sections,  165. 
Inulin,  375. 

"       method  of  testing,  376. 
Iodine  alcohol,  266. 

"       and  cellulose,  318-323. 

"       early  use  of,  267. 

"       and  glycerine,  286. 

"         "    lignin,  331. 

"       potassium  iodide  of,  286. 

"       solutions  of,  285. 

"  "          kept  in  the  dark,  288. 

«       and  starch,  360. 

"       water,  285. 
Iron  plate  for  grinding  sections,  209. 

J. 

Janssen,  Hans  and  Zacharias,  inventors 

of  compound  microscope,  3. 
Jones,  7. 


K. 

King,  Rev.  J.  D.,  Providence  microtome, 

189. 
King's  amber  cement,  235. 

"       lacquer  finish,  236. 

"       method  of  sealing  cells  by  heat,  244. 

"       fluid  for  marine  algae,  225. 

"       white  cement,  236. 
Knife  for  cutting  sections,  195. 
Koch's  embedding  medium,  186. 
Kyanophyll,  412. 

spectrum  of,  417. 

L. 

Labelling  preparations,  245. 

Laboratory  table,  253. 

Lancets  and  needles,  171, 172. 

Leeuwenhoek's  microscopes,  2. 

Lens  holder,  103, 104. 

Lichenin,  368,  371. 

Life  cells,  making,  251. 

Light,  regulating  the,  for  drawing,  N.,  116. 

Lignin,  317,  330. 

"       and  aniline  sulphate,  333. 

"         "    indol,  338. 

"        "    iodine  reagents,  331. 


Lignin  and  phenol-muriatic  acid,  340. 

"         "    phloroglucin,  336. 

"         reactions,  relative  sensitiveness 
of,  342. 

Living  objects,  examination  of,  249. 
Lonicera,  staining  sections  of,  268,  269. 
Lyccena,  scale  dots  of,  57,  58. 


Maceration  of  plants,  162. 

"          "       "    by  boiling,  162. 
Machines  for  making  rock  sections,  207, 

210. 

Magnification,  the  highest,  66. 
•'       "       measuring,  44. 
"       "       produced   by  objective,  44, 
"        "       tables  of,  45,  46,  47,  48. 
Manipulation  in  preparing  reagents,  273. 
Marginal  rays,  26. 

Markings  on  butterflies'  scales,  58,  61. 
Marsh's  bleaching  process,  201. 
Martin,  7. 
Mason,  N.  N.,  originator  of  Providence 

microtome,  188. 

Mastic  varnish  for  cement,  234. 
Matter  (Stoffe),  the  term  how  used,  315. 
Measuring  cylinder,  275. 
14       flask,  274. 
"       by  camera  lucida,  127. 
Mercuric  chloride,  295. 

"    nitrate,  296. 
Metaplasm,  396. 
Methyl-green  aniline,  301. 

"       "       for  cell  nucleus,  401. 
"      violet  aniline,  301. 
Method  of  aniline  sulphate  reaction  on 

wood,  334. 
Method  of  cleaning  diatoms,  430. 

"       "     investigating  asparagin,  426, 
427. 

Method  of  iodine  reactions  on  wood,  333. 
"       "  phenol-muriatic  acid  reaction, 
341. 

Method  of  phloroglucin  reaction,  336. 
*'       "  testing  asaron,  441. 

cane  sugar,  378. 
dextrine,  365. 
grape  sugar,  377. 
resin, 444. 
tannin,  437,  438. 
Micro-chemistry,  not  a  valid  term,  269. 
Micrometer,  the  cob-web,  127. 
"          the  ocular,  122. 
"       ocular-glass,  122-3. 
"  "    screw,  126. 

"    objective,  121. 
"          "       glass,  121. 
"          "       screw,  121. 


INDEX. 


461 


Micrometric  tables,  131. 
Micrometry,  121. 

"       in  general,  128-133. 
•'       unit  in,  130. 
Micron  unit  in  microraetry,  130. 
Microscope  accessories  for  the,  100. 
"       botanical  dissecting,  107. 
"       compact  dissecting,  106. 
'«       the  compound  described,  14. 
««          «          »       invention  of,  3. 

«          "       dissecting,  107, 108. 
"        foot,  95,  96. 

"       magnifying  power  of  early,  5. 
«  "  "       "  modern,  43. 

"       handy  dissecting,  103. 
"       histological,  dissecting,  108. 
"       history  and  name  of,  1. 
"       how  to  judge  a,  53. 
»•       the  mounting,  100. 
u       stand  described,  15. 
•«  •'    the  Acme,  18. 

"          "    Biological,  21. 

"    Histological,  20. 
"  "    Illustrator's,  20. 

"  "    Model,  17. 

"  "    Physicians',  18. 

"          "    New  Student,  18. 
"  "    Student,  16,  19. 

"          "    Universal,  21. 
"    testing  optical  powers  of,  53. 
*•    tube,  71. 

"       "    like  telescope,  5. 
"       use  ol  to  be  learned, 9. 
"  ««    "    Sachs  on,  10. 

u  u    "    rules  for,  96-99. 

Microscopic  image  drawing,  110. 

"  "    projection  of,  110,  112. 

"  "    reflected    by   mirror, 

113. 

Microscopic  image  reflected  by  prism,  111. 
"  '•    requisites  for  produc- 

ing, 15. 
Microscopic  preparations  of  fossil  plants, 

203. 
Microscopic      preparations,      historical 

sketch  of,  157. 
Microscopic  preparations  should  always 

be  transparent,  157. 
Microscopic  sections,  cutting,  177. 
Microscopical  analysis,  2(38. 

«•  '•    founded  by  Hartig' 

270. 

Microscopical  analysis  not  concluded,  270. 
"        measuring,  120. 

reagents,  267,  268. 

"  '•       an   experimental  sci- 

ence, 270. 

Microscopical  technique,  156. 
Microscopist,  personal  qualities  of,  12. 


Micro-spectroscope,  139. 

"  "       the  binocular,  143. 

«'  *<       description  of,  140-3. 

«*  "       measuring  apparatus  of, 

144. 

Microtome,  Caldwell  automatic,  N.,  195. 
"       the  common,  188. 
"         «•    Providence,  189. 
**         "    Taylor  freezing,  191-3. 
"         "    Thoma  sliding,  N.,  195. 
Middle  lamella,  317,  343. 

"          "    its  pectose  metamorphos   c. 
346. 

Middle  lamella,  how  produced,  344. 
"          "    reactions  on,  345. 
"    layer,  56. 
Milk-saps,  446. 

"    of  Euphorbia,  446. 
Millon's  reagent,  296. 

Mineral  acids,  reactions  on, cellulose,  323. 
Mirror,  the,  82,  83. 

••        "    use  of,  83,  84. 
«•       "    introduced   by  Culpeper  and 
Scarlet,  4. 

Modifications  of  cellulose,  315,  318. 
Mohl,  Hugo  v,  8. 

"         "    on  microscopic  preparations, 
157. 

Mohl,  Hugo  v,  section  making,  165. 
Mohr's  spring  compressor  burette,  277. 
Moist  chamber,  251,  253. 
Molecular  intercalation,  269. 
Monobrom-naphihaline     lor      mounting 

262. 
Mounting  fluid,  217. 

"  "    for  algae,  225. 

"  "    Canada  balsam,  222. 

•«  "    chloride  of  calcium,  223. 

"  "    a  colored,  226. 

"  "    corrosive  sublimate,  224. 

"  "    creasote  mixture,  224. 

"  "    glycerine,  218. 

"    jelly,  220. 

•*  "    gum  mastic  and  dammar, 

223. 

Mounting  fluid  monobrom-napthaline,  226. 
"  "    potassium  acetate,  -2'25. 

"    in  fluid,  process  of,  228,  229. 
"    fluid  Styrax and  liquid  amber,  226. 
"       "    sugar  water,  224. 
"       "    table  of  refraction  of,  159. 
"        "    Topping's,  225. 
Mucilage,  characteristic,  36S..369. 
"    of  flax,  374. 
"    vegetable,  367. 
Mucilaginous  cellulose,  327. 

"  "    reactions  for,  329. 

Muculent  cellulose,  317. 
Muriatic  acid,  284. 


462 


INDEX. 


N. 

Nachet's  binocular  ocular,  39,  41. 
Nageli  and  iodine  reagents,  270. 
Naturalist,  the,  must  use  simple  tools,  165. 
Needles  and  lancets,  171, 172. 
Neubauer's  cuprammonia,  294. 
Neutral  tint  reflector,  115. 
Nicol,  W.,  first  ground  sections  of  fossil 

wood,  206. 
Nicol's  prism,  134. 
Nitric  acid,  284. 

"       "    and  alcohol  for  bleaching,  202. 
Nitrogenous  combinations,  314. 
Nitzschia  linearis,  68. 
Nobert's  camera  lucida,  117. 

•'    test  plate,  70. 
Nose  piece,  the  Congress,  75. 
«    Facility,  75. 
«    Fasoldt,76, 
"         "       "    Triple,  74. 
"         "       "    Zentmayer,  75. 
Nucleus  of  the  cell,  397. 

"        stain  ing  the,  268, 399,  403. 
"        structure  of  the,  398. 


O. 

Objects  always  mounted  in  fluids,  158. 
Objects  for  immediate  observation,  160. 
Object  table,  80. 
44       slides,  214. 

"          «'       different  forms  of,  215. 
Objective,  the,  22. 

"       condenser,  94. 

"       micrometer,  121. 

"       glass-micrometer,  121. 

"       protector,  31. 

"       screw-micrometer,  121. 

4'       system, 23,  29. 

"  "       requirements  of,  24. 

"  "       screw  collar  adjustment 

of,  34. 

Objectives,  table  of  American,  50,  51. 
Observation  by  artificial  light,  94,  95. 
Ocular,  Divini's,  4. 

"       Huygenian,  35-38. 

"       Kellner's.  38. 

4>       llamsden's,  38. 

"       glass-micrometer,  122. 

"          "  "          use  of,  124-5. 

"       screw-micrometer,  126. 

"       micrometer    used   iu    drawing, 
254,  255. 

Oil  crayons  for  coloring  drawings,  266. 
"    stones  for  razors,  168. 
Oils,  essential,  441. 
"    fatty,  440. 

Opaque  objects  not  used,  158. 
Optical  powers,  testing  the,  53. 


Osmic  acid,  296. 
Over-corrected  lenses,  28. 
Oxalic  acid,  298. 

"          "    standard  solution  of,  278. 


P. 

Palmellin,450. 

Paraffins  and  tallow  for  embedding,  186, 
187. 

Parallel  compressor,  176. 

Parnassus  pahistris,  hairs  of,  264. 

Pectose   metamorphosis    of   the  middle 
lamella,  346. 

Permanent  preparations,  157. 

making,  213. 

Petrified  woods,  treatment  of,  205. 

Pleffer's  method  of  making  proteid  grains 
insoluble,  383. 

Pfitzer's  reagent  for  hardening  and  stain- 
ing cell  nucleus,  402. 

Phenol,  302. 

Phenol-muriatic  acid  and  lignin,  340. 

Phloroglucin,  302. 

"  and  lignin,  336. 

Phosphoric  acid,  285. 

Photo-micrography,  119. 

Phykocyan,  450. 

•'          spectrum  of,  452. 

Phykoerythrin,  450. 

PhykophaBin,  450. 

Phykoxanthin,  449. 

"  spectrum  of,  452. 

Physical  reactions,  268,  269. 

Physiological    functions    of  asparagin, 
426. 

Physiological  functions  of  silicates,  429. 

Picro-anilme,  301. 

Picro-carmine,  309. 
"  "  for  cell  nucleus,  400. 

"       haematoxylin  for  cell  nucleus,  400. 

Pieris  brassicce,  scale  of,  59. 

Pillsbury  cabinet,  249. 

Pilobulus,  crystalloids  in,  388. 

Pinularia  nobilis  and  viridis,  62. 
4i         viridis,  drawing,  255. 

Pipette  for  handling  unicellular  plants, 
161, 162. 

Pipette  for  measuring,  274, 275. 

Plant  substances  of  limited  distribution, 
431. 

Plant  ^substances  of  universal  distribu- 
tion, 313. 

Plasmodium  of  Myxomycetae,  396. 

Plossl,  8. 

Pleurosigma  angulatum,  64,  69. 

with  high  pow- 
ers, 65. 


INDEX. 


463 


Pleurosigma  angulatum,  low  powers,  64. 
«  "       medium    "      65. 

«  balticum,  63. 

"          species  lor  test  objects,  63. 
Polarizing  apparatus,  133, 134. 

<•  use  of,  136. 

Porcelain  crucible,  174. 

"         dishes,  173. 

Potassium  acetate,  for  mounting,  225. 
alcohol,  290. 

"          for  bleaching,  200. 
"  bichromate,  291. 

»'  chlorate,  291. 

"          copper-sulphate    and    cellu- 
lose, 325,  326.     * 
Potassium  ferrocyanide,  298. 
"  hydroxide,  288. 

t,  "  bottles  for,  289. 

«<  "  for  clarUying  sec- 

tions, 199. 
Potassium  hydroxide,  preserving,  289. 

«  •»        as  a  tauniu  reagent, 

437. 
Potassium  nitrate,  291. 

"         standard  solution  of,  279. 
"         solution  of  definite  concentra- 
tion, 282. 
Potassium,  to  find  percentage  of  solution, 

281. 
Potash,  caustic,  preparation  of  solution, 

288,  289. 

Potato  tuber  crystalloids,  387. 
Poulsen,  author  of  micro-chemistry,  271. 
Poulsen's  shellac  cement,  235. 
Preparation  of  microscopic  objects,  156. 

"          of  objects   without   cutting, 
160. 

Preparation  of  permanent  mounts,  213. 
Preparations  clarified  with  glycerine,  198. 

cataloguing,  246, 247. 
«  labelling,  245. 

"  storing,  247-9. 

Preparing  microscope,  100. 
Preserving  fluid,  carbolic  acid,  227. 
«'  "    for  fungi,  222. 

.«  "      "    lichens.  227. 

"  "    Wickersheimer's,  228. 

"          media.  -217. 
Primordial  utricle  shrinks  in  glycerine, 

158. 

Prism,  illuminating,  89. 
"    the  Nicol's,  134. 
"    polarizing  and  analyzing,  135. 
Proteids,  379. 
Proteid  grains,  amoi-phic,  382. 

"    coloring  with  alcanna,  385. 
<«  "    with  crystalloids,  385,  391. 

"  "    discovered  by  Hartig,  381. 

"          "    with  globoids,  391. 


Proteid  grains  inclosing  inorganic  matter, 

390. 

Proteid  grains  made  insoluble,  383. 
"    matter,  functional,  392. 
"  "    with  starch,  363. 

Protoplasm,  392,  393. 
"    drawing.  2G2. 
"    in  the  narrow  sense,  394. 
"    reactions  of,  395. 
Providence  microtome,  189. 
Prussiate  of  potash,  298. 
Pteris  aquilina,  section  of  rhizome,  257. 
"          "    fibro-vascalar    bundles,    in 
polarized  light,  136-7. 


Rack  and  pinion,  7. 

"    for  holding  rock  sections,  213. 
Radlkofer's  chlor-iodide  of  zinc,  287. 
Razor  for  cutting  sections,  166. 
"    different  forms  of,  166. 
<k    honing,  169, 
"    how  to  sharpen,  168, 170. 
"    the  J.  R.  Torrey  flattened,  167. 
"    strops,  the  J.  R.  Torrey,  169. 
Reactions  of  cell  contents,  table  of  403 
"  "    cell  nucleus,  397. 

"          of  chlorophyll  coloring  matter, 
410. 

Reactions  of  essential  oils,  441. 
'«  '•    fatty  oils,  440. 

milk-saps,  446. 
mucilaginous  cellulose,  329. 
proteid  grains,  384. 
protoplasm,  395. 
resin,  443,  444. 
"       table  of  cellulose,  356. 
"        of  tannin,  437,  438. 
"         "  wax,  440, ' 
Reagent  bottle,  272. 
Reagents,  preparing,  273. 

microscopical,  267,  268. 
"       volumetric  method  of  preparing, 

278. 

Refraction,  exponent  of,  158. 
Refractive  index  of  fluids,  table  of,  159. 

"    power  of  fluids,  value  of  in  investi- 
gations, 159. 
Removing  the  air  from  the  section,  196, 

197. 

Reserve  proteid  substances,  380. 
Resin,  442. 

"    betula,  445. 

"    drops  stained  with  alcanna,  310. 
"    meal,  443,  444. 
"        "    test  for,  444,  445. 
"    production  of,  443. 
Resolving  power,  44. 

•«  "    testing  the,  57. 


464 


INDEX. 


Rhodospermin,  389. 

Kiddell,  J.  L.,  inventor  of  binocular  mic- 
roscope, 39. 
Rock  sections  grinding  down  ,  208. 

"  "  "    thin,  209. 

"          "    labelling,  212. 

"  "    machines  lor  making,  207, 210. 

"  "    mounting,  211. 

"  "    preserving,  212,  213. 

Rubber  cement,  Brown's,  238. 
Jtiubia  tinctorum,  coloring  matter  of,  447. 
Russow's  bleaching  process,  200. 
Rules  for  the  use  of  the  microscope,  96. 


S. 


s'  method  of  incineration,  164. 
Sachs  on  microscopical  drawing,  11. 

"'      "  microscopical  preparations,  156. 

"      "    use  of  microscope,  10. 
Salicin,  433. 
Salix  purpurea,    contains  phloroglucin, 

302. 

Sambucus  nigra  elder  pith,  183. 
Scalpels,  171. 

'•    different  forms  of,  170. 

"    sharpening,  171. 
Schultze's  maceration  mixture,  163. 

'•       mixture  and  cork  tissue,  352. 

"       warming  stage,  82. 
Schweigger-Seidel  acid  carmine  solution, 

308. 

Schweitzer's  reagent,  293. 
Scissors  and  lorceps,  172. 
Screw-collar  adjustment,  shown,  34. 
Screwing  ou  the  objective,  98. 
Sealing  cells  with  heat,  244. 
Section  cutting  with  cork,  184. 

"          "    .       "%  elder  pith,  183. 

"    in  embedding  media,  185. 

"  "    free  hand,  179. 

"  "    longitudinal,  181,  182. 

"    with  a  microtome,  187. 

"  "    transverse,  180. 

"    further  handling  of,  196. 

"    now  to  handle,  181. 

"    instrument,  165. 

"    knife,  195. 

"    lifter,  196. 
'    machines  the  common,  188. 

"    removing  air  from,  196-7. 
'    under  preparing  microscope,  197. 
Sections,  cutting  microscopic,  177. 

"    of  fossil  wood,  'ground,  20(5. 
Self-knowledge  of  the  microscopist,  13. 
Sharpening  razors,  168,  169,  170. 

"    scalpels,  171. 
Shellac  cement,  Foulsen's,  235. 

"          **    Thiersch's,  235. 


Shellac  and  sealing  wax  cement,  234. 

Silex,  430. 

Silicious  skeleton  of  plants,  430. 

Simplex,  the.100. 

Single-celled  plants,  161. 

Slides,  how  to  clean,  216. 

Slit,  influence  of,  ou  spectrum,  151-2. 

Society  screw,  71,  73. 

Sodium  chloride,  291. 

"    nuro-prusbiate,  298. 
Solanum  Americanuiti,  crystalloids  in,  390. 
Solar  energy  and  chlorophyll,  421. 
Sorby's  tubes  for  the  uiicro-speotroscope, 

101. 

Specific  gravity  of  fluids,  278. 
Spectra,  absorption,  153. 

"    of  chlorophyll,  415. 
Spectroscopic  analysis  ot  alga;,  45u,  451, 

"    behavior  of  chlorophyll,  413. 
Spectrum  of  Anihoxanthm,  42ii-i. 
"    etiohu,  418,  4ia. 
"    Jbloridia  green ,  451. 
*'  "    leaf,  41(3,  417. 

"  "    phycocyau,  452. 

kt    phycoxanthm,  452. 
Spherical  aberration,  5. 
Spiral  spring  clip,  116. 

"    tissue,  drawing,  2(52. 
Spirit  lamp  and  tripod,  175. 
Spirogyra,  Beale's  carmine  lor,  308. 
Spring  clip,  spiral,  17U. 
Stage,  7,  80. 
"       the  circular,  81. 
"        mechanical,  81. 
"        warming,  si,  82. 
Staining  the  nucleus,  399. 
Stand,  the  early,  of  wood,  7. 
*'        of  brass,,  8. 

the  microscope,  15. 
Standard  potassium  solution,  279. 
•'        solution  of  oxalic  acid,  278. 

"        "     sulphuric  acid,  280. 
tables    of    equivalents 
for,  281. 

Standard  scale,  148. 
Starch,  357,  359. 

"    in  chlorophyll  grains,  364. 
"    grams,  drawing,  263. 
"    colored  by  iodine,  267. 
"    two  elements  of,  362. 
"    and  iodine  reagents,  360. 
"    first  visible  product  of  assimila- 
tion, 358. 

Starch  in  proteid  plasmic  matter,  363. 
"    reactions  of,  360. 
"    solubility  of,  362. 
"    and  tannin,  436. 
Stearoptene,  441. 
Stepheuson's  binocular  ocular,  40. 


INDEX. 


465 


Storing  permanent  preparations,  247-219. 

Strops,  razor,  the  J.  R.  Torrey,  169. 

Styrax  and  liquidamber  in  mounting,  226. 

Suberin,  318,  347. 

Sugar,  cane,  378. 
"    grape,  377. 

Sulphuric  acid,  284. 

"          "    to  dilate  to  20  per  cent  so- 
lution, 282. 

Sulphuric    acid   and  iodine  reaction  on 
cellulose,  323,  324. 

Sulphuric  acid,  standard  solution  of,  280. 

Surirella  gemma,  66,  68. 

Synapta,  anchors  of,  55. 
"       plates  of,  54. 

Syracuse  watch  glass,  174. 

Syringin,  434. 


T. 

Table  of  American  objectives,  50,  51. 
"       "    apertures,  Prof.  Abbe. 
"       "    cellulose  reactions,  356. 
"       "  comparison  of  English  and  met- 
ric scales,  opp.  p.  133. 

Table  of  equivalents  for  standard  solu- 
tions, 281. 

Table  of   magnification,  of  Bausch  and 
Lomb  lenses,  48. 

Table  of  magnification  of  Hartnack  lens- 
es, 45. 

Table  of  magnification  of  various  combi- 
nations, 47. 

Table  of  magnification  of  strongest  lens- 
es, 46. 

Table  of  magnification  of  lenses  of  vari- 
ous makers,  46. 

Table,  micrometric,  131. 
"      of  reactions  on  cell  contents,  403. 
"        "  refraction  of  fluids,  159. 
"        "  specific  gravity  of  fluids,  278. 

"  test  objects,  70. 
"       "  vegetable  substances,  314. 
a  laboratory,  253. 

Tannic  acid,  435. 

Tannin,  435. 

"      as  a  chromogen,  436. 
"      reactions  of,  437,  438. 
"      and  starch,  436. 
"      tested  by  ferric  oxide,  268. 

Taxus  baccata,  drawing  cells  of,  260. 

Taylor,  the  freezing  microtome,  191. 

"        how     to 

use,  193. 
Taylor,  the  freezing   microtome,  modifi- 

cation  of,  194, 195. 
Technique,  microscopical,  156. 


30 


Test  objects,  53,  71. 

"       •'  table  of,  70. 

Testing  the  defining  power,  54. 

"        "    resolving  power,  57. 
Thalophytes  tested  with  indol,  339. 
Thiersch's  borax  carmine,  307. 

"  oxalic  acid  carmine,  307. 

"  shellac  cement,  235. 

Thoma  sliding  microtome,  N.,  495. 
Tissue  colorless  in  natural  state,  267. 
"       epidermal   and   aniline   sulphate, 
335. 
Tissue  epidermal  and  indol,  339. 

"       of  vascular  bundles  and  indol,  340. 
Tolles'  binocular  ocular,  39. 
Topping's  fluid  for  mounting,  225. 
Torrey's  flattened  razor,  167. 

"       razor  strop,  169. 
Tradescantia,  circulation  of,  protoplasm 

in,  160. 

Triple  nose-piece,  74. 
Trommer's  copper  sulphate  and  potash 

test, 377. 
Turn-table,   self-centering,   Bausch    and 

Lomb's,  240. 
Turn-table,self-centering,  Beck's,  239. 

Hervey's,  241. 
"          Zentmayer'8,240. 
use   of   in   making   cement 
cells,  231. 

Turn-table,  use  of  in  mounting,  242,  243. 
Turpentine,  442. 


U. 

Under-corrected  lenses,  28. 

Unit  in  micrometry,  130. 

Units  of  measure,  comparison  table  of. 

131. 

Universal  accessory,  90,  91. 
Use  of  the  burette,  277. 


V. 

Van  Deyl,  made  first  achromatic  micro- 
scope, 6. 

Vanillin,  433. 

Vascular  bundles  of  Lonicera,  269. 

"       tested  by  aniline    sul- 
phate, 335. 

Vascular  plants  tested  with  indol,  339. 

Vegetable  substances,   classification   of, 
312,  313. 

Vegetable  substances  of  universal  occur- 
rence, 313,  314. 

Vegetable  mucilage,  367. 

Veratrum,  438. 

Volumetric     method    in    preparing    re. 
agents,  278. 


466 


INDEX. 


W. 


Walmsley's   photo-micrographic  appara- 
tus, 120. 

Ward's  eye  shade,  43. 
"       hand  rest,  108. 
"       iris  illuminator,  91. 
Warming  the  microscope  in  winter,  99. 
Wash  bottle,  175. 
Watchglass,  the  Syracuse,  174. 
"    glasses,  combination  of,  174. 
"       "         a  set  of,  174. 
Water,  283. 

•'  colors  for  drawings,  265. 
Watman  paper  for  drawing,  266. 
Wax,  440. 

"    cells  for  mounting,  238. 
"    cement,  234. 
Weber  life  slide,  259. 
Webster  condenser,  89. 
Weigert's  picro-carmine,  310. 
Weighing  scales,  274. 
Wenham's  binocular  ocular,  40,  41. 

"          button  condenser,  88. 
White  zinc  cement,  238. 


Wickcrsheimer's  fluid,  228. 
Wiesner's  cuprammonia,  294. 
Wolf,  4. 

Wollaston's  camera  lucida,  115,  116. 
Wood,  bituminous,  205. 

"       cells,  drawing,  261. 

"       cellulose,  317. 

"       petrified,  205. 

"       substance,  330. 


Xanthein,  422. 
Xanthin,  422. 
Xanthophyll,  412,  41S-. 

"  spectrum  of,  419. 


Zeiss'  binocular  ocular,  41. 
Zentmayer's  nose-piece,  75. 
Zirkel,  an  early  German  petrologist,  206. 


EKRATA,  ETC. 


Page  19,  line  8,  before  A  insert  ].  Page  32,  line  20,  for  J  read  T.  Page 
43,  Fig.  17  is  printed  upside  down.  Page  52,  line  11,  for  opposite  read  oQth. 
Page  91,  in  Fig.  31,  the  lower  middle  figure  should  show  a  black  center-stop 
instead  of  a  central  aperture.  Page  92,  line  7,  for  if,  is  hoped  will  soon  be, 
read  has  lately  been.  Page  108*,  line  4,  for  cm  read  mm.  Page  109,  line  6 
from  bottom,  after  The  insert  dotted.  Pages  156-7  for  Microscopical  objects 
and  preparations  read  Microscopic,  etc.  Page  158,  2nd  paragraph,  10th  line, 
read  primordial  utricle.  Page  282,  first  line,  read,  "  To  prepare  a  potash 
solution  of  a  definite  concentration."  Page  296,  2nd  paragraph,  2nd  line, 
read  fuming  for  foaming.  Page  307,  5th  line  from  bottom,  for  Beal's  read 
Beale's.  Page  350,  9th  line  from  top,  read  fall  for  falls. 

Headline  of  section,  p.  121,  for  I  read  1.  Headline  p.  126,  for  B.  read  3. 
Headline,  p.  127,  for  III  read  IV.  Headline,  p.  128,  for  IV  read  V.  Headline 
p.  133,  for  IV  read  VI.  Headline  p.  139,  for  V  read  VII.  Headline  p.  177, 
for  Microscopical  read  Microscopic. 

The  metric  system  of  weights  and  measures  is  used  throughout  in  this 
work.  The  degrees  of  temperature  are  from  the  centigrade  thermometer. 


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ACID,  ACETIC  C.  P. 

ACID,  CARBOLIC  C.  P. 

ACID,  CHROMIC  C.  P. 

ACID,  HYDROCHLORIC  C.  P. 

ACID.  NITRIC  C.  P. 

ACID,   OXALIC  C.  P. 

ACID,  SULPHURIC  C.  P. 

ACID,  OSMIC 

ACID,  PICRIC 

ACID,  PYROGALLIC  C.  P. 

A  LC  ANN  IN 

ALCOHOL,  ABSOLUTE 

ALDEHYDE  C.  P. 

ALIZARIN 

ALUM,  POTASSIC  C.  P. 

ALUM  CARMINE 

AMMONIUM  CARMINATE 

ANILIN,  C.  P. 

ANILIN  COLORS,  Soluble 

ANTHOCYANIN 

ASPHALT  CEMENT 

A  SPA  U  AGIN 

BALSAM,  CANADA,  Filtered 

BALSAM  IN  BENZOLE 

BALSAM  IN  CHLOROFORM 

BENZOLE,  Pure 

BRUCINE 

BRUNSWICK  BLACK 

BUTTER,  COCOA,  Filtered 

CADMIUM  CHLORIDE 

CARBON  BISULPHIDE 

CARMINE  No.  40 

SOLIE 


CARMINE  SOLUTION 

CHRYSAROBIN 

CHLOROFORM  C.  P. 

CLOVE  OIL,  Pure 

COTOIN 

DAMAR  IN  TURPENTINE 

DlPHENYLAMIN 

ETHER,  PURIFIED 

GELATINE 

GLYCERINE,  ENGLISH 

GLYCERINE  JELLY 

GLYCOGEN 

GOLD  CHLORIDE 

ILEMATOXYLIN 

HOMOGENEOUS  IMMERSION  FLUIDS 

INDIGOTIN 

INDOL 

IODINE  RESUB 

NAPHTHALINE  MONOBROMIDE 

NAPHTHALINE 

PHLOROGLUCIN 

POTASSIC  HYDRATE  C.  P. 

ROSOLIC  ACID 

RESORCIN 

SUBERIN 

TANNIN  C.  P.,  Soluble 

THYMOL 

TOLUIDIN 

TY  ROSIN 

VANILLIN 

XANTHEIN 

ZINC  CIILOUIODIDE. 

ZEHOIR, 


Becker  Bros,  Balances  and  Weights  of  Precision 

Orders  for  Microscopes  and  Objectives  promptly  filled 
at  manufacturer's  prices, 


J.  T.  BROWN. 


J.  T.  BROWN,  Jr. 


C.  H.  BASSETT. 


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